Materials and Methods for Producing Alkaloids

ABSTRACT

The invention generally relates to methods of producing loline alkaloids or precursors thereof, expression constructs, and host cells useful for producing loline alkaloids or precursors thereof, and methods for producing loline alkaloids or precursors thereof in a host cell.

FIELD OF THE INVENTION

The present invention relates to materials and methods of producing loline alkaloids or precursors thereof.

BACKGROUND TO THE INVENTION

Loline alkaloids are produced symbiotically during infection of grasses by Epichloe species fungal endophytes. These endophytes are considered to be bioprotective, conferring pest, and possibly drought and disease protection to the symbionts of which they form part.

Lolines are potent broad spectrum insecticidal alkaloids with no observed toxicity to animals. These fungal secondary metabolites are major contributors to the bioprotective pest tolerance conferred on cool season grasses by Epichloë endopytes.

A robust and/or scalable method for preparing loline alkaloids in the absence of a symbiotic relationship is not presently available.

There is a need for a method to produce loline alkaloids in fungi that do not natively produce lolines in order to extract and use lolines as a natural pesticide.

It is an object of the present invention to provide improved materials and methods for producing loline alkaloids or precursors thereof, and/or at least provide the public with a useful choice.

SUMMARY OF THE INVENTION Host Cell

In one aspect, the invention provides a host cell modified or transformed to comprise at least one polynucleotide selected from the group consisting of:

-   -   i) a polynucleotide encoding a polypeptide comprising the         sequence of any one of SEQ ID NO:1, 12, 13 and 61 or a variant         thereof with at least 40% identity to any one of SEQ ID NO:1,         12, 13 and 61 with at least one of activity of a gamma-class PLP         enzyme and an activity substantially equivalent to that of a         lolC gene product,     -   ii) a polynucleotide encoding a polypeptide comprising the         sequence of any one of SEQ ID NO:2, 14, 15 and 62 or a variant         thereof with at least 40% identity to any one of SEQ ID NO:2,         14, 15 and 62 with at least one of activity of an alpha-class         PLP enzyme and activity substantially equivalent to that of the         lolD gene product,     -   iii) a polynucleotide encoding a polypeptide comprising the         sequence of any one of SEQ ID NO:3, 16, 17 and 63 or a variant         thereof with at least 40% identity to of any one of SEQ ID NO:3,         16, 17 and 63 with at least one of monooxygenase activity and         activity substantially equivalent to that of the lolF gene         product,     -   iv) a polynucleotide encoding a polypeptide comprising the         sequence of any one of SEQ ID NO:4, 18 and 19 or a variant         thereof with at least 40% identity of any one of SEQ ID NO:4, 18         and 19 with at least one of amino acid bridging activity and         activity substantially equivalent to that of the lolA gene         product,     -   v) a polynucleotide encoding a polypeptide comprising the         sequence of any one of SEQ ID NO:5, 20, 21 and 64 or a variant         thereof with at least 40% identity to of any one of SEQ ID NO:5,         20, 21 and 64 with at least one of activity of an alpha-class         PLP enzyme and activity substantially equivalent to that of the         lolT gene product,     -   vi) a polynucleotide encoding a polypeptide comprising the         sequence of of any one of SEQ ID NO:6, 22, 23 and 65 or a         variant thereof with at least 40% identity to of any one of SEQ         ID NO:6, 22, 23 and 65 with at least one of activity of a         non-heme iron dioxygenase and activity substantially equivalent         to that of the lolE gene product,     -   vii) a polynucleotide encoding a polypeptide comprising the         sequence of any one of SEQ ID NO:7, 24, 25 and 66 or a variant         thereof with at least 40% identity to any one of SEQ ID NO:7,         24, 25 and 66 with at least one of activity of a non-heme iron         dioxygenase and activity substantially equivalent to that of the         lolO gene product,     -   viii) a polynucleotide encoding a polypeptide comprising the         sequence of any one of SEQ ID NO:8, 26 and 27 or a variant         thereof with at least 40% identity to any one of SEQ ID NO:8, 26         and 27 with activity substantially equivalent to that of the         lolU gene product,     -   ix) a polynucleotide encoding a polypeptide comprising the         sequence of SEQ ID NO:9 or 28 or a variant thereof with at least         40% identity to SEQ ID NO:9 or 28 with at least one of         N-Methyltransferase activity and activity substantially         equivalent to that of the lolM gene product,     -   x) a polynucleotide encoding a polypeptide comprising the         sequence of any one of SEQ ID NO:10, 29 and 67 or a variant         thereof with at least 40% identity to any one of SEQ ID NO:10,         29 and 67 with at least one of acetamidase activity and activity         substantially equivalent to that of the lolN gene product, and     -   xi) a polynucleotide encoding a polypeptide comprising the         sequence of SEQ ID NO:11 or 30 or a variant thereof with at         least 40% identity to SEQ ID NO:11 or 30 with at least one of         cytochrome P450 monooxygenase activity and activity         substantially equivalent to that of the lolP gene product.

In one embodiment the host cell is modified or transformed to comprise at least 2, preferably at least 3, more preferably at least 4, more preferably at least 5, more preferably at least 6, more preferably at least 7, more preferably at least 8, more preferably at least 9, more preferably at least 10, more preferably at least 11 polynucleotides selected from i) to xi)

In a further embodiment the host cell is modified or transformed to comprise the polynucleotides of i), ii), iii), v) and vii). That is the host cell is modified or transformed to comprise a lolC, lolF, lolD, lolT and a lolO gene, or polynucleotides encoding a lolC, lolF, lolD, lolT and a lolO gene product.

In a further embodiment the host cell contains more copies of the at least one polynucleotide than does a control cell.

In a further embodiment the host cell is not modified or transformed to comprise the polynucleotide of vi). That is the host cell is not modified or transformed to comprise a lolE gene, or a polynucleotide encoding a lolE gene product.

In a further embodiment the host cell is not modified or transformed to comprise the polynucleotide of viii). That is the host cell is not modified or transformed to comprise a lolU gene, or a polynucleotide encoding a lolU gene product.

In a further embodiment the host cell is not modified or transformed to comprise the polynucleotides of vi) or viii). That is the host cell is not modified or transformed to comprise a lolE or a lolU gene, or polynucleotides encoding a lolE or a lolU gene product.

In one embodiment the host cell produces more of at least one loline alkaloid or precursor thereof, than does a control cell.

In a further embodiment the host cell produces more of at least one loline alkaloid or precursor thereof, than does a control cell, as a result of the host cell being transformed or modified to comprise at least one polynucleotide.

In a further embodiment the control cell has not been modified or transformed to comprise the at least one polynucleotide.

In a further embodiment the control cell is of the same species or strain as the host cell that has been modified or transformed to comprise the at least one polynucleotide

Host Cell where Polynucleotide is Part of an Expression Construct

In a further embodiment the at least one polynucleotide is part of an expression construct.

In a further embodiment the at least one polynucleotide is operably linked to a promoter.

In a further embodiment the at least one polynucleotide is operably linked to a terminator.

In a further embodiment the at least one polynucleotide is operably linked to a promoter and a terminator.

Expression Construct

In a further aspect, the invention provides an expression construct comprising at least one polynucleotide selected from the group consisting of:

-   -   i) a polynucleotide encoding a polypeptide comprising the         sequence of any one of SEQ ID NO:1, 12, 13 and 61 or a variant         thereof with at least 40% identity to any one of SEQ ID NO:1,         12, 13 and 61 with at least one of activity of a gamma-class PLP         enzyme and an activity substantially equivalent to that of a         lolC gene product,     -   ii) a polynucleotide encoding a polypeptide comprising the         sequence of any one of SEQ ID NO:2, 14, 15 and 62 or a variant         thereof with at least 40% identity to any one of SEQ ID NO:2,         14, 15 and 62 with at least one of activity of an alpha-class         PLP enzyme and activity substantially equivalent to that of the         lolD gene product,     -   iii) a polynucleotide encoding a polypeptide comprising the         sequence of any one of SEQ ID NO:3, 16, 17 and 63 or a variant         thereof with at least 40% identity to of any one of SEQ ID NO:3,         16, 17 and 63 with at least one of monooxygenase activity and         activity substantially equivalent to that of the lolF gene         product,     -   iv) a polynucleotide encoding a polypeptide comprising the         sequence of any one of SEQ ID NO:4, 18 and 19 or a variant         thereof with at least 40% identity of any one of SEQ ID NO:4, 18         and 19 with at least one of amino acid bridging activity and         activity substantially equivalent to that of the lolA gene         product,     -   v) a polynucleotide encoding a polypeptide comprising the         sequence of any one of SEQ ID NO:5, 20, 21 and 64 or a variant         thereof with at least 40% identity to of any one of SEQ ID NO:5,         20, 21 and 64 with at least one of activity of an alpha-class         PLP enzyme and activity substantially equivalent to that of the         lolT gene product,     -   vi) a polynucleotide encoding a polypeptide comprising the         sequence of of any one of SEQ ID NO:6, 22, 23 and 65 or a         variant thereof with at least 40% identity to of any one of SEQ         ID NO:6, 22, 23 and 65 with at least one of activity of a         non-heme iron dioxygenase and activity substantially equivalent         to that of the lolE gene product,     -   vii) a polynucleotide encoding a polypeptide comprising the         sequence of any one of SEQ ID NO:7, 24, 25 and 66 or a variant         thereof with at least 40% identity to any one of SEQ ID NO:7,         24, 25 and 66 with at least one of activity of a non-heme iron         dioxygenase and activity substantially equivalent to that of the         lolO gene product,     -   viii) a polynucleotide encoding a polypeptide comprising the         sequence of any one of SEQ ID NO:8, 26 and 27 or a variant         thereof with at least 40% identity to any one of SEQ ID NO:8, 26         and 27 with activity substantially equivalent to that of the         lolU gene product,     -   ix) a polynucleotide encoding a polypeptide comprising the         sequence of SEQ ID NO:9 or 28 or a variant thereof with at least         40% identity to SEQ ID NO:9 or 28 with at least one of         N-Methyltransferase activity and activity substantially         equivalent to that of the lolM gene product,     -   x) a polynucleotide encoding a polypeptide comprising the         sequence of any one of SEQ ID NO:10, 29 and 67 or a variant         thereof with at least 40% identity to any one of SEQ ID NO:10,         29 and 67 with at least one of acetamidase activity and activity         substantially equivalent to that of the lolN gene product, and     -   xi) a polynucleotide encoding a polypeptide comprising the         sequence of SEQ ID NO:11 or 30 or a variant thereof with at         least 40% identity to SEQ ID NO:11 or 30 with at least one of         Cytochrome P450 monooxygenase activity and activity         substantially equivalent to that of the lolP gene product.

In one embodiment the expression comprises at least 2, preferably at least 3, more preferably at least 4, more preferably at least 5, more preferably at least 6, more preferably at least 7, more preferably at least 8, more preferably at least 9, more preferably at least 10, more preferably at least 11 polynucleotides selected from i) to xi).

In a further embodiment the expression construct comprises at least the polynucleotides of i), ii), iii), v) and vii). That is the construct comprises at least a lolC, lolF, lolD, lolT and a lolO gene, or at least polynucleotides encoding a lolC, lolF, lolD, lolT and a lolO gene product.

In a further embodiment the at least one polynucleotide is operably linked to at least one promoter.

In a further embodiment the expression construct does not comprise the polynucleotide of vi). That is the expression construct does not comprise a lolE gene, or a polynucleotide encoding a lolE gene product.

In a further embodiment the expression construct does not comprise the polynucleotide of viii). That is the expression construct does not comprise a lolU gene, or a polynucleotide encoding a lolU gene product.

In a further embodiment the expression construct does not comprise the polynucleotides of vi) or viii). That is the expression construct does not comprise a lolE or a lolU gene, or polynucleotides encoding a lolE or a lolU gene product.

Host Cell Comprising the Construct

In a further aspect the invention provides a host cell comprising at least one construct of the invention.

Those skilled in the art will understand that the desired complement of lol genes or polynucleotides may be present in one or multiple constructs that are transformed into the host.

In a preferred embodiment the at least one polynucleotide, or expression construct, is stably incorporated into the genome of the host cell.

Host Cell is Tolerant of ACPP Production

In a further embodiment the host cell is tolerant of endogenous (3-amino-3-carboxypropyl)proline (ACPP) production.

In a further embodiment the host cell has been pre-selected for tolerance to said level of cellular ACPP.

In a further embodiment the host cell has been pre-selected for tolerance of endogenous ACPP production by transformation with at least one polynucleotide encoding a polypeptide comprising the sequence of SEQ ID NO:1 or a variant thereof with at least 40% identity to SEQ ID NO:1 with at least one of activity of a gamma-class PLP enzyme and activity substantially equivalent to that of a lolC gene product.

In a further embodiment the host cell was supplied, or fed, with O-acetyl-L-homoserine (OAH) during selection. Preferably the host cell was supplied, or fed, with non-limiting amounts of OAH during selection.

In a further embodiment the host cell was supplied, or fed, with L-proline during selection. Preferably the host cell was supplied, or fed, with non-limiting amounts of L-proline during selection.

In a further embodiment the host cell is tolerant to at least 0.2 mM, more preferably at least 0.4 mM, more preferably at least 0.6 mM, more preferably at least 0.8 mM, more preferably at least 1 mM, more preferably at least 1.2 mM, more preferably at least 1.4 mM, more preferably at least 1.6 mM, more preferably at least 1.8 mM, more preferably at least 2 mM, more preferably at least 2.2 mM, more preferably at least 2.4 mM, more preferably at least 2.6 mM, more preferably at least 2.8 mM, more preferably at least 3 mM, more preferably at least 3.2 mM, more preferably at least 3.4 mM, more preferably at least 3.6 mM, more preferably at least 3.8 mM, more preferably at least 4 mM, more preferably at least 4.2 mM, more preferably at least 4.4 mM, more preferably at least 4.6 mM, more preferably at least 4.8 mM, more preferably at least 5 mM, more preferably at least 5.2 mM, more preferably at least 5.4 mM, more preferably at least 5.6 mM, more preferably at least 5.8 mM, more preferably at least 6 mM, more preferably at least 6.2 mM, more preferably at least 6.4 mM, more preferably at least 6.6 mM, more preferably at least 6.8 mM, more preferably at least 7 mM, more preferably at least 7.2 mM, more preferably at least 7.4 mM, more preferably at least 7.6 mM, more preferably at least 7.8 mM, more preferably at least 8 mM ACPP in the growth medium.

In a further embodiment host cell is tolerant of a level of cellular ACPP that is toxic to a control cell of the same strain or species.

Host Cell can Convert 1-AP to AcAP

In a further embodiment the host cell, prior to modification or transformation, is able to convert exo-1-aminopyrrolizidine (1-AP) to exo-1-acetamido-pyrrolizidine (AcAP).

In a further embodiment the host cell, prior to modification or transformation, has been pre-selected for the ability to convert 1-AP to AcAP.

In a further embodiment the host cell has been pre-selected by measuring AcAP production in the host cell.

In a further embodiment the host cell was supplied, or fed, with 1-AP during selection. Preferably the host cell was supplied, or fed, with non-limiting amounts 1-AP during selection.

In a further embodiment the selected host cell can produce at least 0.005 milligrams (mg), preferably 0.01 milligrams (mg), more preferably 0.02 milligrams (mg), more preferably 0.03 milligrams (mg), more preferably 0.04 milligrams (mg), 0.05 milligrams (mg), more preferably at least 0.1 mg, more preferably at least 0.15 mg, more preferably at least 0.2 mg, more preferably at least 0.25 mg, more preferably at least 0.3 mg, more preferably at least 0.35 mg, more preferably at least 0.4 mg, more preferably at least 0.45 mg, more preferably at least 0.5 mg, more preferably at least 0.75 mg, more preferably at least 1 mg, more preferably at least 1.5 mg, more preferably at least 2 mg of AcAP per gram (g) of cellular biomass.

Host Cell Type

In a further embodiment the cell is from a fungal species.

In a further embodiment the cell is from a bacterial species.

In a further embodiment the cell is from the subkingdom Dikarya.

In a further embodiment the cell is from a phylum selected from Chytridiomycota, Neocallimastigomycota, Blastocladiomycota, Glomeromycota, Ascomycota and Basidiomycota or a subphylum incertae sedis selected from Mucoromycotina, Kickxellomycotina, Zoopagomycotina and Entomophthoromycotina.

In a further embodiment the cell is from an order selected from Mucorales, Hypocreales, Eurotiales, Sebacinales and Saccharomycetales.

In a further embodiment the cell is from a genus selected from Metarhizium, Epichloë, Saccharomyces, Kluveromyces, Trichoderma, Aspergillus, Beauveria, Pichia, Penicillium, Serendipita, Umbelopsis, Neurospora, Epicoccum, Sarocladium, Balansia, Fusarium, Alternaria, Ustilago, Sebacina, Glomus and Rhizopus.

In a further embodiment the cell is from the species Metarhizium robertsii. In a further embodiment the cell is from the species Trichoderma reesei. In a further embodiment the cell is from the species Aspergillus niger. In a further embodiment the cell is from the species Aspergillus nidulans. In a further embodiment the cell is from the species Aspergillus oryzae. In a further embodiment the cell is from the species Beauveria bassiana. In a further embodiment the cell is from the species Saccharomyces cerevisiae. In a further embodiment the cell is from the species Pichia pastoris. In a further embodiment the cell is from the species Kluveromyces marxianus. In a further embodiment the cell is from the species Epichloë festucae. In a further embodiment the cell is from the species Epichloë typhina. In a further embodiment the cell is from the species Penicllium chrysogenum. In a further embodiment the cell is from the species Penicillium paxilli. In a further embodiment the cell is from the species Penicillium expansum. In a further embodiment the cell is from the species Serendipita indica. In a further embodiment the cell is from the species Umbelopsis isabellina. In a further embodiment the cell is from the species Neurospora crassa. In a further embodiment the cell is from the species Epicoccum italicum. In a further embodiment the cell is from the species Sarocladium zeae. In a further embodiment the cell is from the species Fusarium verticillioides. In a further embodiment the cell is from the species Ustilago maydis.

In one embodiment, the cell is a fungal cell other than a yeast cell.

In one embodiment the cell is a yeast cell.

In a further embodiment the cell is from a non-Epichloë uncinata fungal species.

Fermentation Suitable Host Cells

In a further embodiment the cell is from species or strain of fungi that is tractable to use in fermentation. In a further embodiment the cell is from a species or strain of fungi capable of a specific growth rate (μh⁻¹) of at least 0.01, preferably 0.02, more preferably at least 0.03, more preferably at least 0.04, more preferably at least 0.05, more preferably at least 0.1, more preferably at least 0.15, more preferably at least 0.2, more preferably at least 0.25, more preferably at least 0.3, more preferably at least 0.35, more preferably at least 0.4, more preferably at least 0.45.

In a one embodiment the host cells suitable for fermentation is from a phylum selected from: Ascomycota and Basidiomycota or subphylum incertae sedis Mucoromycotina. In one embodiment the host cells suitable for fermentation are from a genus selected from: Aspergillus, Beauveria, Epichloë, Neurospora, Epicoccum, Sarocladium, Kluveromyces, Metarhizium, Penicillium, Pichia, Rhizopus, Saccharomyces, Serendipita, Trichoderma, and Umbelopsis.

In a one embodiment the host cells suitable for fermentation is from a species selected from: Aspergillus niger, Aspergillus nidulans, Aspergillus oryzae, Beauveria bassiana, Epichloe festucae, Epichloë typhina, Epicoccum italicum, Metarhizium robertsii, Penicillium expansum, Penicillium chrysogenum, Penicillium paxilli, Saccharomyces cerevisiae, Kluveromyces marxianus, Pichia pastorus, Rhizopus oryzae, Rhizopus stolonifer, Rhizopus microsporus, Serendipita indica, Trichoderma reesei, Neurospora crassa, Sarocladium zeae and Umbelopsis isabellina.

Method for Producing a Host Cell for Producing at Least One Loline Alkaloid, or Precursor Thereof

In a further aspect the invention provides a method for producing a host cell for producing at least one loline alkaloid or precursor thereof, the method comprising modifying or transforming a host cell to comprise at least one polynucleotide as herein described.

In a further embodiment the host cell is produced by transforming a cell to comprise at least one polynucleotide or construct as herein described.

Method for Producing at Least One Loline Alkaloid or a Precursor Thereof

In a further aspect the invention provides a method for producing at least one loline alkaloid or a precursor thereof, the method comprising culturing host cells of the invention, or produced by a method of the invention, under conditions conducive to the production of the at least one loline alkaloid or precursor thereof, by the host cells.

In one embodiment the method further comprises separating, purifying, fractionating or isolating the at least one loline alkaloid or precursor thereof.

In a further embodiment the host cells are cultured in the presence of at least one loline alkaloid precursor.

In various embodiments the method comprises maintaining the host cells in the presence of at least one of:

-   -   (a) an effective amount of proline or a biosynthetic precursor         thereof,     -   (b) an effective amount of O-acetyl-L-homoserine (OAH) or a         biosynthetic precursor thereof,     -   (c) an effective amount of (3-amino-3-carboxypropyl)proline         (ACPP) or a biosynthetic precursor thereof,     -   (d) an effective amount of exo-1-aminopyrrolizidine (1-AP) or a         biosynthetic precursor thereof,     -   (e) an effective amount of exo-1-acetamido-pyrrolizidine (AcAP)         or a biosynthetic precursor thereof, or     -   (f) any combination of two or more of (a) to (e) above.

In one embodiment the method comprises maintaining the host cells, or a culture thereof, at a temperature of from about 15° C. to about 35° C.

In a further embodiment the method comprises maintaining the host cells, or a culture thereof, at a temperature of from about 15° C. to about 40° C.

In one embodiment the method comprises maintaining the host cells, or a culture thereof, at for at least about 1 day, at least about 3 days, at least about 4 days, at least about 7 days or at least about 10 days.

In one embodiment the method comprises maintaining the host cells, or a culture thereof, in a bioreactor.

In one exemplary embodiment the method is a method of producing one or more loline alkaloids, comprising:

-   -   i) providing a culture comprising a host cell of the invention,     -   ii) maintaining the culture for at least about 1 day at a         temperature of from about 15° C. to about 40° C. in the presence         of one or more of the following:         -   (a) an effective amount of proline or a biosynthetic             precursor thereof,         -   (b) an effective amount of O-acetyl-L-homoserine (OAH) or a             biosynthetic precursor thereof,         -   (c) an effective amount of (3-amino-3-carboxypropyl)proline             (ACPP) or a biosynthetic precursor thereof,         -   (d) an effective amount of exo-1-aminopyrrolizidine (1-AP)             or a biosynthetic precursor thereof,         -   (e) an effective amount of exo-1-acetamido-pyrrolizidine             (AcAP) or a biosynthetic precursor thereof, or         -   (f) any combination of two or more of (a) to (e) above, and     -   iii) separating the one or more loline alkaloids from the         culture, or at least partially purifying or isolating the one or         more loline alkaloids, thereby to provide the one or more loline         alkaloids.

In one embodiment the purification or isolation is achieved via filtration and/or column purification.

In one aspect the invention provides a method for conferring the ability to produce a loline alkaloid or a precursor thereof on an organism, the method comprising transforming the organism with an expression construct of the invention.

In one embodiment the organism, prior to transformation, does not produce the loline alkaloid or precursor thereof.

The cell as herein described may be part of an organism. Thus reference to a cell or host cell can be used interchangeably with reference to an organism or host organism.

In one embodiment the loline alkaloid or precursor thereof is toxic to a pest.

In one embodiment the pest is an insect.

The term “insect” includes, but not limited to, aphids, mealybugs, whiteflies, moths, butterflies, psyllids, thrips, stink bugs, rootworms, weevils, leafhoppers and fruit flies, such as Myzus persicae (green peach aphid), Aphis gossypii Glover (melon/cotton aphid), Rhopalosiphum maidis (corn leaf aphid), Aphis glycines Matsumura (soybean aphid), Brevicoryne brassicae (cabbage aphid), Anasa tristis (squash bug); Pseudococcus longispinus (long tailed mealybug), Pseudococcus calceolariae (scarlet mealybug), Pseudococcus viburni (obscure mealybug), Planococcus citri (Citrus mealybug); Trialeurodes vaporariorum (greenhouse whitefly), Bemisia tabaci (silverleaf whitefly); Plutella xylostella (diamondback moth), Citripestis sagittiferella (citrus fruit moth), Helicoverpa armigera (tomato fruitworm or corn earworm), Pectinophora gossypiella (pink bollworm), Phthorimaea operculella (potato tuber moth), Amyelois transitella (Navel orangeworm), Cydia pomonella (codling moth), Cnephasia jactatana (black-lyre leafroller), Epiphyas postvittana (light-brown apple moth), Grapholita molesta (oriental fruit moth), Ostrinia furnacalis (Asian corn borer), Ostrinia nubilalis (European corn borer), Scirpophaga excerptalis (sugarcane top borer) Diatraea saccharalis (sugarcane borer), Chilo plejadellus (rice stalk borer), Earias vitella (spotted bollworm), Earias insulana (spiny bollworm), Spodoptera frugiperda (fall armyworm), Spodoptera litura (tobacco cutworm), Melittia cucurbitae (squash vine borer), Teia anartoides (painted apple moth), Trichoplusia ni (Cabbage looper); Pieris rapae (white butterfly); Bactericera cockerelli (tomato/potato psyllid), Diaphorina citri (Asian citrus psyllid), Trioza erytreae (African citrus psyllid); Thrips obscuratus (flower thrips), Heliothrips haemorrhoidalis (greenhouse thrips), Thrips tabaci (onion thrips), Frankliniella williamsi (Maize thrip); Halyomorpha halys (brown marmorated stink bug), Oebalus pugnax (rice stink bug), Diabrotica virgifera virgifera (western corn rootworm), Diabrotica barberi (northern corn rootworm), Diabrotica undecimpunctata howardi (southern corn rootworm), Diabrotica virgifera zeae (Mexican corn rootworm); Pempheres affinis (cotton stem weevil); Nephotettix virescens (green leafhopper), Nilaparvata lugens (brown planthopper); Bactrocera tryoni (Queensland fruit fly).

In one embodiment the pest is a non-insect pest.

In one embodiment the pest is a nematode.

The term “nematode” includes but is not limited to root-knot nematodes (Meloidogyne species), cyst nematodes (Heterodera and Globodera species), lesion nematodes (Pratylenchus species), reniform nematodes (Rotylenchulus reniformis), lance nematodes (Hoplolaimus species) and stem and bulb nematodes (Ditylenchus species).

Preferred LOL genes for use in various aspects and embodiments of the invention are lolC, lolD, lolF, lolT and lolO.

In certain embodiments the host cells and organisms are not transformed to express lolE. In certain embodiments the host cells and organisms and are not transformed to express lolU. In certain embodiments the host cells and organisms are not transformed to express lolE or lolU.

Any one or more of the following embodiments may relate to any of the aspects described herein or any combination thereof.

In will be appreciated that the polynucleotide may be an allelic variant, degenerate sequence, homologue or orthologue of the specified nucleotide sequences.

In various embodiments the variant polypeptide has at least 40%, more preferably at least 41%, more preferably at least 42%, more preferably at least 43%, more preferably at least 44%, more preferably at least 45%, more preferably at least 46%, more preferably at least 47%, more preferably at least 48%, more preferably at least 49%, more preferably at least 50%, more preferably at least 51%, more preferably at least 52%, more preferably at least 53%, more preferably at least 54%, more preferably at least 55%, more preferably at least 56%, more preferably at least 57%, more preferably at least 58%, more preferably at least 59%, more preferably at least 60%, more preferably at least 61%, more preferably at least 62%, more preferably at least 63%, more preferably at least 64%, more preferably at least 65%, more preferably at least 66%, more preferably at least 67%, more preferably at least 68%, more preferably at least 69%, more preferably at least 70%, more preferably at least 71%, more preferably at least 72%, more preferably at least 73%, more preferably at least 74%, more preferably at least 75%, more preferably at least 76%, more preferably at least 77%, more preferably at least 78%, more preferably at least 79%, more preferably at least 80%, more preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% amino acid identity with the specified polypeptide sequences.

In various embodiments the polynucleotide variant comprises one or more alternative codons that code for the eventual translation of a polypeptide having at least 40%, more preferably at least 41%, more preferably at least 42%, more preferably at least 43%, more preferably at least 44%, more preferably at least 45%, more preferably at least 46%, more preferably at least 47%, more preferably at least 48%, more preferably at least 49%, more preferably at least 50%, more preferably at least 51%, more preferably at least 52%, more preferably at least 53%, more preferably at least 54%, more preferably at least 55%, more preferably at least 56%, more preferably at least 57%, more preferably at least 58%, more preferably at least 59%, more preferably at least 60%, more preferably at least 61%, more preferably at least 62%, more preferably at least 63%, more preferably at least 64%, more preferably at least 65%, more preferably at least 66%, more preferably at least 67%, more preferably at least 68%, more preferably at least 69%, more preferably at least 70%, more preferably at least 71%, more preferably at least 72%, more preferably at least 73%, more preferably at least 74%, more preferably at least 75%, more preferably at least 76%, more preferably at least 77%, more preferably at least 78%, more preferably at least 79%, more preferably at least 80%, more preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% amino acid identity with the specified polypeptide sequences.

In various embodiments the polynucleotide variant having at least 40%, more preferably at least 41%, more preferably at least 42%, more preferably at least 43%, more preferably at least 44%, more preferably at least 45%, more preferably at least 46%, more preferably at least 47%, more preferably at least 48%, more preferably at least 49%, more preferably at least 50%, more preferably at least 51%, more preferably at least 52%, more preferably at least 53%, more preferably at least 54%, more preferably at least 55%, more preferably at least 56%, more preferably at least 57%, more preferably at least 58%, more preferably at least 59%, more preferably at least 60%, more preferably at least 61%, more preferably at least 62%, more preferably at least 63%, more preferably at least 64%, more preferably at least 65%, more preferably at least 66%, more preferably at least 67%, more preferably at least 68%, more preferably at least 69%, more preferably at least 70%, more preferably at least 71%, more preferably at least 72%, more preferably at least 73%, more preferably at least 74%, more preferably at least 75%, more preferably at least 76%, more preferably at least 77%, more preferably at least 78%, more preferably at least 79%, more preferably at least 80%, more preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% nucleotide sequence identity to the specified polynucleotide sequence.

Polynucleotide and polypeptide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polynucleotide sequences using global sequence alignment programs for e.g., the Needleman-Wunsch global alignment program (Needleman and Wunsch, 1970). A full implementation of the Needleman-Wunsch global alignment algorithm is found in the needle program in the EMBOSS package (Rice, et al., 2000) which can be obtained from the World Wide Web at http://www.hgmp.mrc.ac.uk/Software/EMBOSS/. The European Bioinformatics Institute server also provides the facility to perform EMBOSS-needle global alignments between two sequences on line at http:/www.ebi.ac.uk/emboss/align/.

Alternatively, the GAP program may be used which computes an optimal global alignment of two sequences without penalizing terminal gaps (Huang, 1994).

A preferred method for calculating polynucleotide and polypeptide % sequence identity is based on aligning sequences to be compared using Clustal X (Jeanmougin, et al., 1998).

In one embodiment the genome of the untransformed (wild type) host cell does not prior to transformation with a construct of the invention comprise the one or more LOL genes. In another embodiment the genome of the untransformed (wild type) host cell does not prior to transformation with a construct of the invention comprise a gene homologous to the one or more LOL genes. In another embodiment the untransformed (wild type) host cell or does not prior to transformation with a construct of the invention express the one or more LOL genes.

In one embodiment the host cell, expression construct or polynucleotide comprises and/or expresses at least the

-   -   a) lolC gene,     -   b) lolC and lolD genes,     -   c) lolC, lolD and lolT genes,     -   d) lolC, lolD, lolT and lolF genes,     -   e) lolC, lolD, lolT, lolF and lolA genes,     -   f) lolC, lolD, lolT, lolF and lolE genes,     -   g) lolC, lolD, lolT, lolF, lolE and lolA genes,     -   h) lolC, lolD, lolT, lolF, and lolO genes,     -   i) lolC, lolD, lolT, lolF, lolA and lolO genes,     -   j) lolC, lolD, lolT, lolF, lolE and lolO genes,     -   k) lolC, lolD, lolT, lolF, lolA, lolE and lolO genes,     -   I) lolC, lolD, lolF, lolT, lolN and lolM genes,     -   m) lolC, lolD, lolF, lolT, lolA, lolN and lolM genes,     -   n) lolC, lolD, lolF, lolT, lolE, lolN and lolM genes,     -   o) lolC, lolD, lolF, lolT, lolE, lolA, lolN and lolM genes,     -   p) lolC, lolD, lolF, lolT, lolN, lolM and lolP genes,     -   q) lolC, lolD, lolF, lolT, lolA, lolN, lolM and lolP genes,     -   r) lolC, lolD, lolF, lolT, lolE, lolN, lolM and lolP genes, or     -   s) lolC, lolD, lolF, lolT, lolA, lolE, lolN, lolM and lolP         genes.     -   t) lolC, lolD, lolF, lolT, lolA, lolE, lolN, lolM, lolP and lolU         genes.

In one embodiment the one or more LOL genes are derived from Epichloë uncinata, Epichloë festucae, Epichloë coenophiala, Epichloë amarillans, Epichloë glyceriae, Epichloë canadensis, Epichloë brachyelytri, Epichloë aotearoae, Epichloë siegelli, Aktinsonella hypoxylon, or Penicillium expansum.

In one embodiment the expression construct, genome or polynucleotide comprises, or the host cell expresses a gene encoding a heterologous acetyltransferase.

In another embodiment the genome of the untransformed (wild type) host cell comprises an endogenous acetyltransferase. In another embodiment the host cell expresses an endogenous acetyltransferase.

Promoters

In one embodiment one or more of the LOL genes are operably linked to a constitutive promoter. In various embodiments the promoter is the histone H3 promoter, the GAPDH promoter, the pna2/tpi hybrid promoter (Aspergillus nidulans or Aspergillus niger), the gpdA promoter (Metarhizium, Aspergillus, or Serendipita), the mbfA promoter, the trpC promoter (Aspergillus nidulans), the hexokinase-1 promoter (Metarhizium robertsii), the class I hydrophobin promoter (Beauveria bassiana) or any other constitutive promoter described herein.

In one embodiment one or more of the LOL genes are operably linked to an inducible promoter. In various embodiments the promoter is the alcA promoter, the alcR promoter, amyB promoter, the gas promoter, the glaA promoter, the niiA promoter, the cbhI promoter, the ctr4 promoter, the thiA promoter or any other inducible promoter described herein.

Those skilled in the art will understand that the different LOL genes may be operably linked to and/or expressed under the control of different promoters and/or terminators.

Loline Alkaloids

In one embodiment the loline alkaloid is selected from the group comprising N-acetylnorloline (NANL), norloline, loline, N-acetylloline (NAL), N-methylloline (NML), N-formylloline (NFL) and a combination of any two or more thereof.

In one embodiment the loline alkaloid or precursor thereof is selected from the group comprising N-acetylnorloline (NANL), norloline, loline, N-acetylloline (NAL), N-methylloline (NML), N-formylloline (NFL), (3-amino-3-carboxypropyl)proline (ACPP), exo-1-aminopyrrolizidine (1-AP), exo-1-acetamido-pyrrolizidine (AcAP), and a combination of any two or more thereof.

In one embodiment the loline alkaloid is selected from the group comprising N-acetylnorloline (NANL), norloline, loline, N-methylloline (NML), N-formylloline (NFL) and a combination of any two or more thereof.

In one embodiment the loline alkaloid or precursor thereof is selected from the group comprising N-acetylnorloline (NANL), norloline, loline, N-methylloline (NML), N-formylloline (NFL), (3-amino-3-carboxypropyl)proline (ACPP), exo-1-aminopyrrolizidine (1-AP), exo-1-acetamido-pyrrolizidine (AcAP), and a combination of any two or more thereof.

Other aspects of the invention may become apparent from the following description which is given by way of example only and with reference to the accompanying drawings.

It is intended that reference to a range of numbers disclosed herein (for example, 1 to 10) also incorporates reference to all rational numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example, 2 to 8, 1.5 to 5.5 and 3.1 to 4.7).

This invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, and any or all combinations of any two or more of said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which this invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.

In this specification, where reference has been made to external sources of information, including patent specifications and other documents, this is generally for the purpose of providing a context for discussing the features of the present invention.

Unless stated otherwise, reference to such sources of information is not to be construed, in any jurisdiction, as an admission that such sources of information are prior art or form part of the common general knowledge in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described by way of example only and with reference to the drawings in which:

FIG. 1 a) shows four transformant M. robertsii ARSEF 23 isolates cultured on M100 medium containing phosphinothricin observed under visible light, b) the same four transformant isolates observed under blue light. Note that isolate no. 1 is not fluorescent while isolates 2, 3, 4 fluoresce green.

FIG. 2 shows an agarose gel photograph showing PCR amplification results with three sets of primers. Labelled in each gel are lanes carrying gDNA of isolates 17, 18, 19, 21, 22, 26, 29, and 30, which gave the correct band size indicating the presence of the Epichloë lolC gene, and the lane with gDNA of isolate 11, which went through the transformation process, but does not carry Epichloë lolC. pPH3-lolC and Epichloe gDNA were used as positive controls for lolC. M. robertsii ARSEF 23 (Mr Ma23) and water were the negative controls.

FIG. 3 shows an agarose gel photograph showing results of the PCR of cDNA of M. robertsii ARSEF 23 (Mr Ma23), transformant isolates and controls. PCR was done on undiluted and 1:10 diluted cDNA. Note the size difference between the band produced by gDNA (330 bp) and cDNA (285 bp).

FIG. 4 (a) shows a typical extracted chromatogram for m/z 217 of M. robertsii ARSEF 23 and isolate 11, a transformant that lacks E. festucae lolC, but carries the same selectable marker as the E. festucae lolC transformants; (b) shows a typical extracted chromatogram for m/z 217 of four transformant isolates, all of which carry E. festucae lolC. Note the presence of the third peak that corresponds to ACPP in (b).

FIG. 5 shows the loline biosynthetic pathway (modified from (Pan, et al., 2014)). Condensation of L-proline and O-acetylhomoserine is the first committed step of the pathway resulting in formation of ACPP. The next chemically detectable intermediates are 1-AP and AcAP. NANL is the first fully cyclized intermediate that will then be converted to the array of lolines in the chemical arsenal against insects. lolU and lolE, genes present in the loline cluster, but with unknown function, are not shown here.

FIG. 6 shows a comparison of chromatograms of chemically-synthesized AcAP standard to the AcAP produced by B. bassiana ‘Bb CT3’ transformant carrying lolCDFA1TEU. Top: chromatogram of AcAP produced by Bb CT3 transformant; bottom: chromatogram of chemically-synthesized AcAP standard. Both chromatograms exhibit the same retention time and transition (169>110), confirming the biologically-produced AcAP is identical to the chemical standard

FIG. 7 shows a comparison of daughter ion scans of chemically-synthesized AcAP standard to the AcAP produced by Bb CT3 transformant. Top: daughter ion scan of chemically-synthesized AcAP standard, bottom: daughter ion scan of AcAP produced by Bb CT3 transformant. While more background noise is present in the biologically-produced AcAP sample due to its relatively low concentration, the scans show the compounds fragment the same, confirming the chemically- and biologically-produced AcAP are the same compound.

FIG. 8 shows a chromatogram of NANL detected in Bb H15, the first heterologous host expressing the first eight genes of the loline production pathway, thus successfully producing NANL, the first fully cyclized intermediate of the loline production pathway.

FIG. 9. Chromatograms of loline pathway intermediates detected in Bb H15 transformant. Top to bottom: Chromatograms showing the peaks corresponding to ACPP, 1-AP, and AcAP. All these intermediates occur prior to NANL, which was also detected in the same sample.

FIG. 10 shows the relative expression of loline genes (vs. actin) in selected B. bassiana transformants. Bb CT3: transformant carrying lolCDFA1TEU, produced AcAP when fed with 2 mM ACPP. No AcAP was observed in SDB+30 mM proline+2 mM OAH, 25K, 4 dpi

(i.e. without added ACPP). BbCT does not carry a functional copy of lolO, thus relative expression of lolO is given as 0.0; Bb H15: transformant carrying lolCDFA1TEOU, produced NANL (0.385 μM) when grown in SDB+30 mM proline+2 mM OAH, 25° C., 4 dpi. Note relatively less expression of lolF and lolO compared to the rest of the loline genes; Bb O16: transformant generated by transforming Bb H15 parent with an additional copy of lolO controlled by the Fl1 histone H3 promoter and glaA terminator. O16 produced NANL (average=1.231 μM) when grown under the same conditions as Bb H15.

FIG. 11 shows a box and whiskers graph of AcAP amount (mg) produced per gram of dry biomass after 72 hours fermentation in presence of 1-AP.

FIG. 12 shows a box and whiskers graph of specific growth rate (h⁻¹) during 1-AP feeding experiment, E. uncinata specific growth rate is provided as a reference.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is in part directed to host cells transformed with, having a genome comprising, or transformed with an expression construct comprising, one or more heterologous LOL genes, and methods of producing such host cells. The invention is also directed to recombinant methods for producing one or more loline alkaloids or precursors thereof by culturing a host cell described herein, and methods for producing, or conferring the ability to produce, one or more loline alkaloids to a host cell or organism. The lolines and precursors there of produced by the host cell, and methods of the invention are useful for controlling pests.

The present inventors have for the first time demonstrated production of lolines from heterologous expression of genes in the lolines biosynthetic pathway. This is the first pyrrolizidine alkaloid to be produced in any heterologous host.

As far as the inventors are aware, although there are numerous publications on lolines and loline genes, there is no publication reporting or even considering the production of lolines in a heterologous host.

While there have been some reports of heterologous expression of individual LOL genes, these studies relate to assessing gene function rather than any attempt to produce lolines.

Loline genes have only been reported in Epichloë, Atkinsonella hypoxylon and Penicillium expansum. In Penicillium and Atkinsonella the products of the LOL gene cluster are only predicted, and the cluster is missing some Epichloë LOL gene equivalents. Thus, there is no evidence to suggest that production of the lolines by Epichloë outside of Epichloë itself is possible.

Furthermore, ACPP, the product of the LoIC enzyme, is reported to be toxic even to the producer fungus, even when applied at relatively low amounts of 4 mM (Faulkner, et al., 2006). Heterologous expression of lolC (performed to attempt to complement a cystathionine synthase mutant) in Aspergillus nidulans was lost after a single subculture (Spiering, et al., 2005). In addition, attempts to express lolC in E. coli were unsuccessful (Schardl, et al., 2007) and the authors state that these results “suggest that lolC or its enzyme product is toxic to cells”.

The toxicity of lolC or its enzyme product, thus makes the applicants successful production of lolines via the expression of LOL genes including lolC all the more surprising. The applicant's invention therefore additionally provides for pre-selection of strains tolerant to ACPP for use in the heterologous production of lolines.

In addition, the applicants have surprisingly shown that none of the reported LOL genes, perform the step of converting 1-AP to AcAP in heterologous hosts in their experiments. However, the applicants have surprisingly shown that this step can be performed by endogenous enzymatic activity present in some strains. The applicant's invention therefore additionally provides pre-selection of strains capable of performing the conversion of 1-AP to AcAP for use in the heterologous production of lolines.

1. Definitions

The term “and/or” can mean “and” or “or”.

The term “comprising” as used in this specification means “consisting at least in part of”. When interpreting statements in this specification which include that term, the features, prefaced by that term in each statement, all need to be present but other features can also be present. Related terms such as “comprise” and “comprised” are to be interpreted in the same manner.

The term “polynucleotide(s),” as used herein, means a single or double-stranded deoxyribonucleotide or ribonucleotide polymer of any length but preferably at least 15 nucleotides, and include as non-limiting examples, genes, coding and non-coding sequences of a gene, sense and antisense sequences complements, exons, introns, genomic DNA, cDNA, pre-mRNA, mRNA, rRNA, siRNA, miRNA, tRNA, ribozymes, recombinant polypeptides, isolated and purified naturally occurring DNA or RNA sequences, synthetic RNA and DNA sequences, nucleic acid probes, primers and fragments. The term also incudes fragments of polypeptides.

A “fragment” of a polynucleotide sequence provided herein is a subsequence of contiguous nucleotides.

The term “polypeptide”, as used herein, encompasses amino acid chains of any length but preferably at least 5 amino acids, including full-length proteins, in which amino acid residues are linked by covalent peptide bonds. Polypeptides of the present invention, or used in the methods of the invention, may be purified natural products, or may be produced partially or wholly using recombinant or synthetic techniques. The term also incudes fragments of polypeptides.

A “fragment” of a polypeptide is a subsequence of the polypeptide that in some embodiments performs a function/activity of and/or influences three-dimensional structure of the polypeptide.

As used herein the term “gene” refers to a polynucleotide sequence or its complement that is involved in producing a polypeptide, including regions preceding (leader) and following (trailer) the coding sequence, and introns between individual coding sequence (exons). It also includes to a codon-optimised polynucleotide sequence of the native gene.

The term “constitutive promoter”, as used herein, refers to a promoter that is not regulated and is active in all conditions in the host cell resulting in continuous transcription of its associated gene.

The term “inducible promoter”, as used herein, refers to a promoter that is regulated and is active in the host cell only in response to specific stimuli resulting in transcription of its associated gene.

A “LOL gene” refers to any of the genes that encode an enzyme involved in catalysing a reaction in the loline biosynthetic pathway, as summarised in FIG. 5 and Table 1 and elsewhere in the specification. This includes any of lolC, lolA, lolT, lolO, lolE, lolN, lolM, lolP, lolU, lolD, or lolF, and/or any of the following polynucleotide sequences: SEQ ID NO. 31 to 60 and 68 to 74. The terms also encompass variants of these polynucleotide sequences as herein defined.

In various embodiment the term “LOL gene” encompasses a polynucleotide selected from the group consisting of:

-   -   i) a polynucleotide encoding a polypeptide comprising the         sequence of any one of SEQ ID NO:1, 12, 13 and 61 or a variant         thereof with at least 40% identity to any one of SEQ ID NO:1,         12, 13 and 61 with at least one of activity of a gamma-class PLP         enzyme and an activity substantially equivalent to that of a         lolC gene product,     -   ii) a polynucleotide encoding a polypeptide comprising the         sequence of any one of SEQ ID NO:2, 14, 15 and 62 or a variant         thereof with at least 40% identity to any one of SEQ ID NO:2,         14, 15 and 62 with at least one of activity of an alpha-class         PLP enzyme and activity substantially equivalent to that of the         lolD gene product,     -   iii) a polynucleotide encoding a polypeptide comprising the         sequence of any one of SEQ ID NO:3, 16, 17 and 63 or a variant         thereof with at least 40% identity to of any one of SEQ ID NO:3,         16, 17 and 63 with at least one of monooxygenase activity and         activity substantially equivalent to that of the lolF gene         product,     -   iv) a polynucleotide encoding a polypeptide comprising the         sequence of any one of SEQ ID NO:4, 18 and 19 or a variant         thereof with at least 40% identity of any one of SEQ ID NO:4, 18         and 19 with at least one of aspartokinase activity and activity         substantially equivalent to that of the lolA gene product,     -   v) a polynucleotide encoding a polypeptide comprising the         sequence of any one of SEQ ID NO:5, 20, 21 and 64 or a variant         thereof with at least 40% identity to of any one of SEQ ID NO:5,         20, 21 and 64 with at least one of activity of an alpha-class         PLP enzyme and activity substantially equivalent to that of the         lolT gene product,     -   vi) a polynucleotide encoding a polypeptide comprising the         sequence of of any one of SEQ ID NO:6, 22, 23 and 65 or a         variant thereof with at least 40% identity to of any one of SEQ         ID NO:6, 22, 23 and 65 with at least one of activity of a         non-heme iron dioxygenase and activity substantially equivalent         to that of the lolE gene product,     -   vii) a polynucleotide encoding a polypeptide comprising the         sequence of any one of SEQ ID NO:7, 24, 25 and 66 or a variant         thereof with at least 40% identity to any one of SEQ ID NO:7,         24, 25 and 66 with at least one of activity of a non-heme iron         dioxygenase and activity substantially equivalent to that of the         lolO gene product,     -   viii) a polynucleotide encoding a polypeptide comprising the         sequence of any one of SEQ ID NO:8, 26 and 27 or a variant         thereof with at least 40% identity to any one of SEQ ID NO:8, 26         and 27 with activity substantially equivalent to that of the         lolU gene product,     -   ix) a polynucleotide encoding a polypeptide comprising the         sequence of SEQ ID NO:9 or 28 or a variant thereof with at least         40% identity to SEQ ID NO:9 or 28 with at least one of         N-Methyltransferase activity and activity substantially         equivalent to that of the lolM gene product,     -   x) a polynucleotide encoding a polypeptide comprising the         sequence of any one of SEQ ID NO:10, 29 and 67 or a variant         thereof with at least 40% identity to any one of SEQ ID NO:10,         29 and 67 with at least one of acetamidase activity and activity         substantially equivalent to that of the lolN gene product, and     -   xi) a polynucleotide encoding a polypeptide comprising the         sequence of SEQ ID NO:11 or 30 or a variant thereof with at         least 40% identity to SEQ ID NO:11 or 30 with at least one of         cytochrome P450 monooxygenase activity and activity         substantially equivalent to that of the lolP gene product.

In one embodiment a lolC gene comprises a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:1, 12, 13 and 61 or a variant thereof with at least 40% identity to any one of SEQ ID NO:1, 12, 13 and 61.

Preferably the lolC gene or variant thereof has at least one of the activity of a gamma-class PLP enzyme encodes and an activity substantially equivalent to that of a lolC gene product.

In a further embodiment the lolD gene comprises a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:2, 14, 15 and 62 or a variant thereof with at least 40% identity to any one of SEQ ID NO:2, 14, 15 and 62.

Preferably the polypeptide or variant thereof has at least one of the activity of an alpha-class PLP enzyme, and an activity substantially equivalent to that of the lolD gene product,

In a further embodiment the lolF gene comprises a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:3, 16, 17 and 63 or a variant thereof with at least 40% identity to of any one of SEQ ID NO:3, 16, 17 and 63.

Preferably the polypeptide or variant thereof has at least one of monooxygenase activity, and activity substantially equivalent to that of the lolF gene product,

In a further embodiment the lolA gene comprises a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:4, 18 and 19 or a variant thereof with at least 40% identity of any one of SEQ ID NO:4, 18 and 19.

Preferably the polypeptide or variant thereof has at least one of amino acid bridging activity and activity substantially equivalent to that of the lolA gene product.

In a further embodiment the lolT gene comprises a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:5, 20, 21 and 64 or a variant thereof with at least 40% identity to of any one of SEQ ID NO:5, 20, 21 and 64.

Preferably the polypeptide or variant thereof has at least one of activity of an alpha-class PLP enzyme and activity substantially equivalent to that of the lolT gene product,

In a further embodiment the lolE gene comprises a polynucleotide encoding a polypeptide comprising the sequence of of any one of SEQ ID NO:6, 22, 23 and 65 or a variant thereof with at least 40% identity to of any one of SEQ ID NO:6, 22, 23 and 65.

Preferably the polypeptide or variant thereof has at least one of activity of a non-heme iron dioxygenase and activity substantially equivalent to that of the lolE gene product,

In a further embodiment the lolO gene comprises a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:7, 24, 25 and 66 or a variant thereof with at least 40% identity to any one of SEQ ID NO:7, 24, 25 and 66.

Preferably the polypeptide or variant thereof has at least one of activity of a non-heme iron dioxygenase and activity substantially equivalent to that of the lolO gene product.

In a further embodiment the lolU gene comprises a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:8, 26 and 27 or a variant thereof with at least 40% identity to any one of SEQ ID NO:8, 26 and 27.

Preferably the polypeptide or variant thereof has activity substantially equivalent to that of the lolU gene product.

In a further embodiment the lolM gene comprises a polynucleotide encoding a polypeptide comprising the sequence of SEQ ID NO:9 or 28 or a variant thereof with at least 40% identity to SEQ ID NO:9 or 28.

Preferably the polypeptide or variant thereof has at least one of N-Methyltransferase activity and activity substantially equivalent to that of the lolM gene product,

In a further embodiment the lolN gene comprises a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:10, 29 and 67 or a variant thereof with at least 40% identity to any one of SEQ ID NO:10, 29 and 67.

Preferably the polypeptide or variant thereof has at least one of acetamidase activity and activity substantially equivalent to that of the lolN gene product.

In a further embodiment the lolP gene comprises a polynucleotide polynucleotide encoding a polypeptide comprising the sequence of SEQ ID NO:11 or 30 or a variant thereof with at least 40% identity to SEQ ID NO:11 or 30.

Preferably the polypeptide or variant thereof has at least one of cytochrome P450 monooxygenase activity and activity substantially equivalent to that of the lolP gene product.

A “LOL gene product” refers to the polypeptide product of any of the genes, i.e. the encoded enzyme, involved in catalysing a reaction in the loline biosynthetic pathway, as summarised in FIG. 5 and Table 1 and elsewhere in the specification. The terms include any of lolC, to/A, lolT, lolO, lolE, lolN, lolM, lolP, lolU, lolD, or lolF polypeptides, and/or any of the following polypeptide sequences: SEQ ID NO. 1 to 30 and 61 to 67. The terms also encompass variants of these polypeptide sequences as herein defined.

The term “heterologous” as used herein with reference to a gene or a polynucleotide or polypeptide sequence transformed into or expressed by a host cell or fungus generally means a gene, or a polynucleotide or polypeptide sequence that is not encoded or expressed naturally by the wild type or native host cell or fungus.

The term “heterologous” as used herein with reference to polynucleotides, promoters and terminators, means that such heterologous sequences are not found operably linked to one another in wild type cells in nature. Thus, for example if a promoter is heterologous to the polynucleotide the promoter and polynucleotide are not found operably linked to one another in wild type cells in nature.

The term “host cell” as used herein refers to a fungal cell line cultured as a unicellular entity, which can be, or has been, used as a recipient for LOL genes, and/or expression constructs bearing one or more LOL genes, and/or which can be, or has been, transformed with or subjected to homologous recombination to integrate one or more heterologous LOL genes into the host cell genome. The term includes the progeny of the original host cell which has been transformed or subjected to homologous recombination. It will be appreciated that the progeny of a parent host cell may not be entirely identical in morphology or in genomic or total DNA complement to the original parent.

The term “plant” as used herein encompasses not only whole plants, but extends to plant parts, cuttings as well as plant products including roots, leaves, flowers, seeds, stems, callus tissue, nuts and fruit, bulbs, tubers, corms, grains, cuttings, root stock, or scions, and includes any plant material whether pre-planting, during growth, and at or post-harvest. Plants that may benefit from the application of the present invention cover a broad range of agricultural and horticultural crops, including crops produced using organic production systems.

The term “plant” includes those from any plant species. Such species include gymnosperm species, angiosperm species, and plants which belong to the superfamily Viridiplantae, in particular monocotyledonous and dicotyledonous plants. Such species include those that are used as fodder or forage crops, ornamental plants, food crops, row crops, horticultural crops, fruit crops, vegetable crops, biofuel crops, timber crops, and other trees or shrubs. Such species may be selected from the following: Acer spp., Actinidia spp., Abelmoschus spp., Agave sisalana, Agropyron spp., Agrostis stolonifera, Allium spp., Amaranthus spp., Ammophila arenaria, Ananas comosus, Annona spp., Apium graveolens, Arachis spp, Artocarpus spp., Asparagus officinalis, Avena spp. (e.g. Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida), Averrhoa carambola, Bambusa sp., Benincasa hispida, Bertholletia excelsea, Beta vulgaris, Brassica spp. (e.g. Brassica napus, Brassica rapa ssp. [canola, oilseed rape, turnip rape, kale]), Cadaba farinosa, Camellia sinensis, Canna indica, Cannabis sativa, Capsicum spp., Carex elata, Carica papaya, Carissa macrocarpa, Carya spp., Carthamus tinctorius, Castanea spp., Ceiba pentandra, Cichorium endivia, Cinnamomum spp., Citrullus lanatus, Citrus spp., Cocos spp., Coffea spp., Colocasia esculenta, Cola spp., Corchorus sp., Coriandrum sativum, Corylus spp., Crataegus spp., Crocus sativus, Cucurbita spp., Cucumis spp., Cynara spp., Daucus carota, Desmodium spp., Dimocarpus longan, Dioscorea spp., Diospyros spp., Echinochloa spp., Elaeis (e.g. Elaeis guineensis, Elaeis oleifera), Eleusine coracana, Eragrostis tef, Erianthus sp., Eriobotrya japonica, Eucalyptus sp., Eugenia uniflora, Fagopyrum spp., Fagus spp., Festuca arundinacea, Ficus carica, Fortunella spp., Fragaria spp., Ginkgo biloba, Glycine spp. (e.g. Glycine max, Soja hispida or Soja max), Gossypium hirsutum, Helianthus spp. (e.g. Helianthus annuus), Hemerocallis fulva, Hibiscus spp., Hordeum spp. (e.g. Hordeum vulgare), Ipomoea batatas, Juglans spp., Lactuca sativa, Lathyrus spp., Lens culinaris, Linum usitatissimum, Litchi chinensis, Lotus spp., Luffa acutangula, Lupinus spp., Luzula sylvatica, Lycopersicon spp. (e.g. Lycopersicon esculentum, Lycopersicon lycopersicum, Lycopersicon pyriforme), Macro tyloma spp., Malus spp., Malpighia emarginata, Mammea americana, Mangifera indica, Manihot spp., Manilkara zapota, Medicago sativa, Melilotus spp., Mentha spp., Miscanthus sinensis, Momordica spp., Morus nigra, Musa spp., Nicotiana spp., Olea spp., Opuntia spp., Ornithopus spp., Oryza spp. (e.g. Oryza sativa, Oryza latifolia), Panicum miliaceum, Panicum virgatum, Passiflora edulis, Pastinaca sativa, Pennisetum sp., Persea spp., Petroselinum crispum, Phalaris arundinacea, Phaseolus spp., Phleum pratense, Phoenix spp., Phragmites australis, Physalis spp., Pinus spp., Pistacia vera, Pisum spp., Poa spp., Populus spp., Prosopis spp., Prunus spp., Psidium spp., Punica granatum, Pyrus communis, Quercus spp., Raphanus sativus, Rheum rhabarbarum, Ribes spp., Ricinus communis, Rubus spp., Saccharum spp., SaNx sp., Sambucus spp., Secale cereale, Sesamum spp., Sinapis sp., Solanum spp. (e.g. Solanum tuberosum, Solanum betaceum, Solanum integrifolium or Solanum lycopersicum), Sorghum bicolor, Spinacia spp., Syzygium spp., Tagetes spp., Tamarindus indica, Theobroma cacao, Trifolium spp., Tripsacum dactyloides, Triticosecale rimpaui, Triticum spp. (e.g. Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum, Triticum monococcum or Triticum vulgare), Tropaeolum minus, Tropaeolum majus, Vaccinium spp., Vicia spp., Vigna spp., Viola odorata, Vitis spp., Zea mays, Zizania palustris, and Ziziphus spp., among others.

The term “loline alkaloid precursor” as used herein refers to compounds produced as intermediates in the loline biosynthetic pathway. Loline alkaloid precursors may comprise the product of reactions catalysed by the enzymatic product of expression of one or more LOL genes.

The term modified, modify, and grammatical variations thereof, with respect to modifying host cells, or fungi to comprise a polynucleotide, include editing the endogenous genome of the host cells or fungi.

Methods for modifying endogenous genomic DNA sequences are known to those skilled in the art. Such methods may involve the use of sequence-specific nucleases that generate targeted double-stranded DNA breaks in genes of interest. Examples of such methods include: zinc finger nucleases (Curtin, et al., 2011, Sander, et al., 2011), transcription activator-like effector nucleases or “TALENs” (Cermak, et al., 2011, Mahfouz, et al., 2011, Li, et al., 2012), and LAGLIDADG homing endonucleases, also termed “meganucleases” (Tzfira, et al., 2012).

Targeted genome editing using engineered nucleases such as clustered, regularly interspaced, short palindromic repeat (CRISPR) technology, is an important new approach for generating RNA-guided nucleases, such as Cas9, with customizable specificities. Genome editing mediated by these nucleases has been used to rapidly, easily and efficiently modify endogenous genes in a wide variety of biomedically important cell types and in organisms that have traditionally been challenging to manipulate genetically. A modified version of the CRISPR-Cas9 system has been developed to recruit heterologous domains that can regulate endogenous gene expression or label specific genomic loci in living cells (Sander and Joung, 2014). The technique is applicable to fungi (Nodvig, et al., 2015).

The term “substantially equivalent” with reference to any given gene product, enzyme, protein, or polypeptide having activity substantially equivalent to that of any given LOL gene product, preferably means that the gene product, enzyme, protein, or polypeptide is capable of fulfilling the role of the LOL gene product as summarised in Table 1 below, and/or has the enzymatic activity listed in Table 1 below.

The term “control”, “controlling”, “biocontrol” or “biological control” are used interchangeably herein to refer to reduction in growth, growth rate, development, feeding rate, reproduction or number of pests, particularly plant pests, and/or reducing the severity of, or eliminating, symptoms of such pests, particularly symptoms in plants caused by such pests.

The term “(s)” following a noun contemplates the singular or plural form, or both.

2. Loline Alkaloid Biosynthesis

Loline alkaloids are produced symbiotically during infection of grasses by endophytes, particularly Epichloë endophytes (which, following a nomenclature realignment, now includes the previously separate anamorph Neotyphodium spp.). These endophytes are considered to be bioprotective, conferring pest, and possibly drought and disease protection to the symbionts of which they form part. Lolines are potent insecticidal compounds and contribute a substantial amount of the bioprotective benefit conferred by Epichloe species that produce them.

Loline alkaloids are 1-aminopyrrolizidines having an oxygen bridge between C2 and C7. The various loline alkaloid variants, namely N-acetylnorloline (NANL), norloline, loline, N-acetylloline (NAL), N-methylloline (NML), N-formylloline (NFL) are differentiated by substituents on the primary amine.

A loline alkaloid biosynthesis pathway has been proposed as shown in FIG. 5.

A loline alkaloid biosynthetic gene cluster has only been identified in fungi belonging to two clades in Pezizomycotina, namely Sordariomycetes (only Epichloë species and Atkinsonella hypoxylon) and Eurotiomycetes (only Penicillium expansum). The Epichloe LOL gene cluster comprises eleven genes, referred to as the LOL genes herein, encoding key enzymes in the loline alkaloid biosynthesis pathway. Homologs to seven of these genes have been reported in P. expansum. The LOL genes are summarised in Table 1 below.

TABLE 1 Summary of LOL genes. Predicted encoded Polypeptide Polynucleotide Gene enzymatic activity Proposed Role SEQ ID NO: SEQ ID NO: Species lolC gamma-class Formation of 1 31 E. festucae PLP ACPP from OAH and proline lolD alpha-class Decarboxylation 2 32 E. festucae PLP of pyrrolodinium ion lolF Monooxygenase Oxidative 3 33 E. festucae decarboxylation of ACPP to form pyrrolodinium ion lolA Amino acid Increasing the 4 34 E. festucae binding levels of OAH lolT alpha-class Cyclisation of 5 35 E. festucae PLP pyrrolodinium ion(s) to form 1-AP lolE Nonheme iron Not clear 6 36 E. festucae dioxygenase lolO Nonheme iron Formation of the 7 37 E. festucae dioxygenase C2-C7 ether bridge in AcAP to form NANL lolU Not clear Not clear 8 38 E. festucae lolM N- Methylation of 9 39 E. festucae Methyl- norloline to form transferase loline, and of loline to form NML lolN Acetamidase Deacetylation of 10 40 E. festucae NANL to form norloline lolP Cytochrome Oxygenation of 11 41 E. festucae P450 NML to form monooxygenase NFL lolC1 gamma-class Formation of 12 42 E. uncinata PLP ACPP from OAH and proline lolC2 gamma-class Formation of 13 43 E. uncinata PLP ACPP from OAH and proline lolD1 alpha-class Decarboxylation 14 44 E. uncinata PLP of pyrrolodinium ion lolD2 alpha-class Decarboxylation 15 45 E. uncinata PLP of pyrrolodinium ion lolF1 Monooxygenase Oxidative 16 46 E. uncinata decarboxylation of ACPP to form pyrrolodinium ion lolF2 Monooxygenase Oxidative 17 47 E. uncinata decarboxylation of ACPP to form pyrrolodinium ion lolA1 Amino acid Increasing the 18 48 E. uncinata binding levels of OAH lolA2 Amino acid Increasing the 19 49 E. uncinata binding levels of OAH lolT1 alpha-class Cyclisation of 20 50 E. uncinata PLP pyrrolodinium ion(s) to form 1-AP lolT2 alpha-class Cyclisation of 21 51 E. uncinata PLP pyrrolodinium ion(s) to form 1-AP lolE1 Nonheme iron Not clear 22 52 E. uncinata dioxygenase lolE2 Nonheme iron Not clear 23 53 E. uncinata dioxygenase lolO1 Nonheme iron Formation of the 24 54 E. uncinata dioxygenase C2-C7 ether bridge in AcAP to form NANL lolO2 Nonheme iron Formation of the 25 55 E. uncinata dioxygenase C2-C7 ether bridge in AcAP to form NANL lolU1 Not clear Not clear 26 56 E. uncinata lolU2 Not clear Not clear 27 57 E. uncinata loIM N-Methyl- Methylation of 28 58 E. uncinata transferase norloline to form loline, and of loline to form NML lolN Acetamidase Deacetylation of 29 59 E. uncinata NANL to form norloline lolP Cytochrome Oxygenation of 30 60 E. uncinata P450 NML to form monooxygenase NFL lolC gamma-class Formation of 61 68 P. expansum PLP ACPP from OAH and proline lolD alpha-class Decarboxylation 62 69 P. expansum PLP of pyrrolodinium ion lolF Monooxygenase Oxidative 63 70 P. expansum decarboxylation of ACPP to form pyrrolodinium ion lolT alpha-class Cyclisation of 64 71 P. expansum PLP pyrrolodinium

3. Expression Constructs and Host Cells

The host cells described herein comprise a genome encoding, expressing or having been transformed with one or more LOL genes or expression construct of the invention.

In various embodiments the expression construct of the invention comprises two or more or three or more LOL genes.

In various embodiments the one or more LOL genes are operably linked to one or more regulatory elements that control the transcription, translation or expression of the gene in a host cell transformed with the expression construct. The one or more regulatory elements may be contiguous with the one or more LOL genes or act in trans or at a distance to control the gene of interest.

Suitable regulatory elements include appropriate transcription initiation, termination, promoter and enhancer sequences, or RNA processing signals such as splicing or polyadenylation signals.

Examples of suitable promoters for use in fungal host cells include promoters which are homologous or heterologous to the host cell. Furthermore, suitable promoters for use in the expression constructs of the invention include constitutive promoters, regulatable promoters, inducible promoters or repressible promoters. The promoter may be derived from a gene of the host cell, or a promoter derived from the genes of other fungi, viruses or bacteria. Those skilled in the art will, without undue experimentation, be able to select promoters that are suitable for use in modifying and modulating expression constructs using genetic constructs comprising the LOL genes of the sequences described herein.

In embodiments where the expression construct comprises two or more LOL genes, or where the host cell comprises or has been transformed with two or more LOL genes, each gene may be under the control of the same promoter or different promoters.

In various embodiments the method comprises transforming the host cell with two or more, three or more, or four or more expression constructs of the invention.

Host cells may be transformed using suitable methods known in the art for achieving heterologous gene expression in fungi and/or yeast. Choice of transformation method will depend on the species and form of the host cell, and the number of expression constructs and or LOL genes to be transformed.

In one embodiment the method comprises transforming the host cell with an expression vector so that the one or more LOL genes is integrated into the genome of the host cell via homologous or non-homologous recombination.

In various embodiments the host cell comprises protoplasts, spheroplasts, spores or conidia.

In one embodiment the method comprises transforming the host cell using polyethylene glycol (PEG)-mediated transformation. Other suitable transformation methods include electroporation, Agrobacterium tumefaciens-mediated transformation, biolistic transformation, or non-PEG-mediated spheroplast transformation.

An exemplary method that may be used to achieve homologous recombination of one or more LOL genes into a fungal host cell genome using sequential transformations is that described by Chiang and co-workers (Chiang, et al., 2013).

Briefly, for genes that are very large and difficult to amplify by PCR, two smaller transforming fragments may be created that fuse by homologous recombination in vivo to reconstruct the full-length coding sequences under the control of a single promoter. Two or more LOL genes may be integrated into the host cell genome using sequential transformations. Each gene or transforming fragment carries a selectable marker to enable selection of transformants that are have been transformed with the gene. Marker recycling may be used so that many genes may be transferred easily into the host cell.

4. Production of Pyrrolizidine Alkaloids In Vitro

Exemplary methods to produce and at least partially purify and/or isolate one or more of the loline alkaloids or of the invention are described herein. These include the at least partial purification and/or isolation of one or more loline alkaloids from a culture of one or more species, or from culture media or culture supernatants and the like obtained therefrom.

In one embodiment the method comprises maintaining a culture of host cells in the presence of one or more loline alkaloid precursors. For example, in various embodiments the culture is maintained in the presence of one or more of

-   -   a) an effective amount of proline or a biosynthetic precursor         thereof,     -   b) an effective amount of O-acetyl-L-homoserine (OAH) or a         biosynthetic precursor thereof,     -   c) an effective amount of (3-amino-3-carboxypropyl)proline         (ACPP) or a biosynthetic precursor thereof,     -   d) an effective amount of aspartic acid or a biosynthetic         precursor thereof,     -   e) an effective amount of exo-1-acetamido-pyrrolizidine (AcAP)         or a biosynthetic precursor thereof,     -   f) an effective amount of exo-1-aminopyrrolizidine (1-AP) or a         biosynthetic precursor thereof, or     -   g) any combination of two or more of (a) to (f) above.

Choice of culture conditions, including duration of culturing, temperature and/or culture media will depend upon the particular characteristics of the host cell.

The invention consists in the foregoing and also envisages constructions of which the following gives examples only and in no way limit the scope thereof.

EXAMPLES Example 1: Heterologous Expression of lolC in Heterologous Hosts to Produce (3-amino-3-carboxypropyl)proline (ACPP) BACKGROUND

The Epichloë LOL gene cluster, consisting of 11 genes, has been reported to be required for loline biosynthesis (Spiering, et al., 2005, Pan, et al., 2014). Epichloë lolC was predicted to encode the enzyme that catalyses the first committed step of the loline pathway—the condensation of L-proline with the 3-amino-3-carboxypropyl group from O-acetyl-L-homoserine (Faulkner, et al., 2006). The biosynthetic intermediate produced by this reaction has a dose-dependent effect in E. uncinata—it is toxic to cells when fed at 4 mM, but results in the enrichment of N-formylloline when fed to cultures at 2 mM. P. expansum, the only species outside the Epichloë Glade known to carry the loline genes, has homologs to Epichloë lolC, D, F, T, E, O, N. There are no published studies characterizing P. expansum lolC (Pe lolC), but based on its similarity to Epichloë lolC, applicants expect that Pe lolC also encodes an enzyme that catalyzes the same reaction as Epichloë lolC (see Table 2 for amino acid percent identity between E. festucae and P. expansum loline gene products).

The applicants considered that heterologous expression of lolC in fungi which do not possess the LOL gene cluster nor produce lolines, is an important first step towards transforming and expressing selected LOL genes in a non-Epichloë fungus, particularly in light of ACPP's reported toxicity. As initial proof of concept, Epichloë festucae lolC (Ef lolC) was expressed in M. robertsii ARSEF 23 and surprisingly the applicants were able to demonstrate that ACPP was produced. This is described in detail below. The applicants further individually expressed Ef lolC and Pe lolC with successful ACPP production in B. bassiana, A. niger, and T. reesei. S. indica was successfully transformed with Pe lolC, the next step will be confirming experession of lolC. A full summary of expression results of lolC and ACPP production in heterologous hosts is given in Table 5.

TABLE 2 Amino acid percent identity between proteins encoded by E. festucae and P. expansum loline genes Gene Amino acid percent identity lolC 63.2% lolD 57.6% lolF 56.9% lolT 61.7% lolE 52.0% lolO 52.5% lolN 45.0%

Protocol

A plasmid carrying E. festucae lolC gene (SEQ ID NO: 31) fused to the constitutively expressed histone H3 promoter (pPH3-loC) and a plasmid carrying phosphinothricin resistance and green fluorescence (pBAR-GFP) were co-transformed into competent protoplasts of M. robertsii strain ARSEF 23. Transformants carrying pBAR-GFP were selected based on their ability to grow on regeneration medium containing phosphinothricin and green fluorescence. Genomic DNA was extracted from the selected transformants, and presence of pPH3-lolC was confirmed through amplification of Ef lolC in three sets of polymerase chain reactions (PCR).

Two parallel approaches were taken in order to analyse Ef lolC expression and activity in M. robertsii. Firstly, RNA was extracted from four transformed M. robertsii isolates carrying Ef lolC, an isolate that was subjected to the transformation process but does not carry lolC, and the parental strain M. robertsii ARSEF 23, with the aim of observing Ef lolC transcription via RNA extraction, cDNA synthesis, and PCR. Secondly, the same strains (above) were grown on M100 medium, freeze-dried, and analysed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for presence of (3-amino-3-carboxypropyl)proline (ACPP), the suggested product of condensation of L-proline and O-acetyl-L-homoserine (OAH), in the transformant mycelium or medium, indicating the activity of the Ef lolC-encoded enzyme.

Results

Co-Transformation and Identification of Ef lolC Transformants

Thirty (30) transformant isolates that exhibited both phosphinothricin resistance and green fluorescence (a representative set of transformants shown in FIG. 1) were obtained from the co-transformation and selection processes.

Three sets of PCRs, which were done using three different primer combinations on genomic DNA (gDNA) of a subset of the transformants isolated above, identified and confirmed transformants carrying the pPH3-lolC (FIG. 2). Of the transformant isolates that gave a positive result indicating presence of the lolC gene in all three PCRs, isolates 17, 18, 21, and 26 were used for RNA extraction and chemical analysis. Parent strain M. robertsii ARSEF 23 and transformant isolate 11, which did not produce a band in all three PCRs were used as negative controls in the same experiments.

Transcription of the Ef lolC Gene

Total RNA extracted from M. robertsii ARSEF 23 and transformant isolates 11, 17, 18, 21, 26 were used for complementary DNA (cDNA) synthesis. In a PCR done using two primers designed to complement two consecutive exons that were separated by an intron, cDNA of transformant isolates 17, 18, 21, 26, produced a 245 bp band, while the pPH3-lolC and gDNA produced a 330 bp band. The size difference between the bands produced by cDNA and gDNA matched the size of the intron, indicating Ef lolC was transcribed to RNA and correctly spliced in transformant isolates 17, 18, 21, and 26. M. Robertsii ARSEF 23 and isolate 11, both not carrying Ef lolC, as well as controls done with water and without any reverse transcriptase (RT) enzyme during cDNA synthesis did not give any bands (FIG. 3).

Chemical Analysis of Ef lolC Transformants

When analysed at m/z 217 (the protonated mass of ACPP), parental strain M. robertsii ARSEF 23 and transformant isolate 11, which did not carry pPH3-lolC, produced two peaks (FIG. 4a ). Transformant isolates 17, 18, 21, and 26, carrying pPH3-lolC, gave three peaks (FIG. 4b ). This result was consistent across all biological replicates of each isolate. The growth medium (M100) and the fungicide Sporekill™ (ICA International Chemicals Ltd., active ingredient Didecyldimethylammonium chloride), which was used to make the cultures non-viable, did not produce any peaks (see Table 3 for a list of peak areas for all samples).

Fragmentation analysis of the third peak present in the chromatograms of Ef lolC-expressing isolates matched those with a compound structurally similar to ACPP. This confirmed that the Ef lolC transcript was translated to its functional enzymatic form, which catalysed the condensation of proline and OAH to produce ACPP.

TABLE 3 Peak areas for the growth medium (M100), fungicide (Sporekill), and three biological replicates each for M. robertsii ARSEF 23 and transformant isolates 11, 17, 18, 21, and 26. RT 2.32 [(3-amino-3- carboxypropyl) RT 0.98 RT 1.51 proline] Sample Peak Area Peak Area Peak Area Sporekill 10% 0 0 0 M100 0 0 0 Ma23-1 1170000 717000 0 Ma23-2 887000 472000 0 Ma23-3 1260000 733000 0 C11-1 1860000 1130000 0 C11-2 2150000 1520000 0 C11-3 1610000 993000 0 C17-1 2060000 1520000 999000 C17-2 1110000 583000 869000 C17-3 1430000 835000 913000 C18-1 1040000 650000 209000 C18-2 1020000 393000 242000 C18-3 1230000 787000 209000 C21-1 1100000 634000 5160000 C21-2 1350000 799000 4920000 C21-3 1120000 660000 5640000 C26-1 1050000 573000 2270000 C26-2 1340000 668000 2430000 C26-3 1790000 1150000 3690000 [Note the inability to quantify the amount of (3-amino-3-carboxypropyl)proline as this analysis was performed in the absence of an authentic standard]

SUMMARY

The E. festucae lolC gene was heterologously expressed in M. robertsii. This was evidenced by the presence of Ef lolC in transformant gDNA, the presence of the correctly-spliced transcript, and the isolation of ACPP—the suggested end product of the reaction catalysed by the enzyme encoded by Ef lolC. This experimental report marks the first-ever documentation of successful stable heterologous expression of lolC in a non-Epichloë fungus, confirms its role in producing ACPP and demonstrates the potential for producing subsequent steps in the loline biosynthetic pathway, which was in doubt due to the previously suggested toxicity of ACPP.

Example 2: Production of Lolines and Loline Intermediates in Heterologous Hosts Introduction

Lolines, which consist of a variable combination of N-acetylnorloline (NANL), N-formylloline (NFL), and N-acetylloline (NAL) in nature, are produced in planta by many endophytic Epichloe species (e.g. E. festucae E2368, E. glyceriae E2772, E. uncinata E167). These alkaloids protect their grass hosts from herbivorous insects, but do not show toxicity towards mammals (Jackson, et al., 1996, Wilkinson, et al., 2000, Schardl, et al., 2013). Blankenship and co-workers (Blankenship, et al., 2001) demonstrated that E. uncinata was able to make lolines in culture, which gave evidence that lolines—and the genes responsible—were of fungal origin. An as yet unidentified plant-encoded acetyl transferase converts loline to NAL, a later ‘decorative’ step that increases the diversity of lolines (Pan, et al., 2014).

In Epichloë, eleven genes (lolC, D, F, A, T, E, O, U, M, N, P) collectively known as the LOL gene cluster, are reported to be required for loline biosynthesis (see FIG. 5 and (Pan, et al., 2014)). Of these, lolC encodes the enzyme that catalyses the first committed step of the loline pathway—the condensation of primary metabolites L-proline and O-acetyl-L-homoserine (OAH), as previously predicted (Faulkner, et al., 2006, Schardl, et al., 2007). However, this was only conclusively demonstrated for the first time in a heterologous host by the applicants in Example 1.

Expression of the eleven genes in the Epichloë LOL cluster was predicted to lead to production of NFL, the end-point of the biosynthetic pathway. Alternatively, eight genes are predicted to be required for production of NANL, which can be converted to an array of lolines. While not predicted to be required for NANL biosynthesis per se, the additional expression of lolA is predicted to increase loline biosynthesis via increasing the biological availability of the precursor molecule OAH.

Outside the Epichloë Glade, the loline genes have only been reported in P. expansum (Ballester, et al., 2015, Marcet-Houben and Gabaldon, 2016), wherein the products of the LOL gene cluster have only been predicted. In addition, the P. expansum LOL gene cluster is missing some Epichloë LOL gene equivalents and has homologs of seven of the Epichloë loline genes (lolC, D, F, T, E, O, N).

Production of NANL in a heterologous host in culture vs. production in Epichloë species, is industrially advantageous because of (1) the ability to use a fast-growing heterologous host in a fermenter for continuous mass production of lolines (vs. very little production per day of lolines in vitro and only during the stationary phase by the slow-growing E. uncinata), and (2) the ability to bias production towards individual loline analogues. Lolines have also proven to be very complex to produce via synthetic chemistry (Faulkner, et al., 2006, Cakmak, et al., 2011), thus favouring production via biological means.

The heterologous hosts tested in the reported experiments are (1) E. festucae Fl1, an Epichloë strain that does not possess the loline cluster; (2) B. bassiana strain K4B1, an insect pathogen which is also fermentation-compatible; (3) A. niger strain ATCC 1015, commonly used for industrial fermentation; (4) T. reesei strain RUT-C30 (ATCC 56765), commonly used for industrial fermentation; (5) M. robertsii ARSEF 23, an insect pathogen; (6) Neurospora crassa, a model fungus commonly used for genetic research; (7) S. cerevisiae, a model fungus commonly used for genetic research and industrial fermentation; (8) S. indica, a plant protective endophyte with a broad Angiosperm host range; and (9) U. isabellina, an endophyte. Fungi listed (1)-(7) belong to phylum Ascomycota, while (8) is a member of the phylum Basidiomycota and (9) is a Mucoromycota.

Materials and Methods Fungal Strains

Strains used in this study and method of genetic transformation are listed in Table 4.

All strains are stored at the Biotelliga laboratory at the University of Auckland, Auckland, New Zealand.

TABLE 4 Fungal strains, media, and transformation methods used in the current study Fungal Method of genetic strain Media used transformation A. niger Wild type and Protoplast transformation (ATCC 1015) hygromycin selection: (standard methods) potato dextrose (PD); Phosphinothricin selection: M100 B. bassiana Wild type: Sabouraud Same as for A. niger K4B1 dextrose (SD); Sulfonyl urea, phosphinothricin, hygromycin selection: Czapeks Dox (CD); Geneticin selection: PD E. festucae Epichloë M100 Fl1 M. robertsii Wild type and ARSEF 23 phosphinothricin selection: M100 N. crassa Wild type: Vogel's Spore electroporation (ICMP 7781) medium N (VM); (Navarro-Sampedro, Phosphinothricin et al., 2007) selection: fructose/glucose/ sorbose (FGS) medium S. cerevisiae Wild type: yeast (Gietz and Schiestl, 2007) BY4743 pepetone dextrose (ATCC 201390) (YPD), G418, uracil auxotroph selection: yeast synthetic defined medium (SD) S. indica Wild type and Protoplast transformation (ATCC 204458) hygromycin selection: (Zuccaro, et al., 2009) and Aspergillus complete Electroporation of medium (ACM); hyphal fragments For growth immediately (Yadav, et al., 2010) prior to and selection after electroporation: Aspergillus minimal medium T. reesei Wild type and Protoplasts generated as RUT-C30 hygromycin selection: described (Penttila, et al., (ATCC 56765) PD; 1987, Gruber, et al., 1990) Phosphinothricin transformed by standard selection: M100 protoplast transformation methods U. isabellina Wild type and (Zhang, et al., 2007) ICMP 22148 hygromycin selection: PD

Genetic Constructs

A detailed list of transformation constructs is given in Table 11. In brief, all transformed Epichloë loline genes (SEQ ID NO:31 to 33 and 35 to 41) were cloned from E. festucae E2368, except for pBTL10 that, in addition to E. festucae lolD and lolF, contains E. uncinata lolA1 coding sequence (from ‘wild type lolA1’—SEQ ID NO 48), pBTL11 that, in addition to E. festucae lolC, lolD and lolF, contains E. uncinata lolA1 coding sequence (from ‘wild type lolA1’—SEQ ID NO 48), pBTL15 that, in addition to E. festucae lolC, contains E. uncinata lolA1 coding sequence (from ‘wild type lolA1’—SEQ ID NO 48), and pBTL57 that contains E. uncinata lolA1 coding sequence (from ‘wild type lolA1’—SEQ ID NO 48). Modified open reading frames [i.e. exons only, codon optimized for Neurospora crassa (using the Codon Optimization Tool at the Integrated DNA Technologies website https://sg.idtdna.com/CodonOpt), and with an HA tag] were also used in some cases. All transformed Penicillium loline genes (SEQ ID NO:68 to 71 and 73) were cloned from P. expansum ICMP 8595. Loline genes were transformed either coupled with a constitutive promoter or with a constitutive promoter and terminator. In a few cases, the gene encoding the selectable marker was present in the same plasmid as the loline genes. In most, however, the appropriate selectable marker was co-transformed with the loline gene constructs. All transformants were selected in media with appropriate selection. PCR screening was done to test for the presence of the loline genes on gDNA preparations done according to standard DNA extraction (miniprep gDNA extraction).

RNA Extraction and qPCR

RNA was extracted from fungal mycelia using the TRIzol® reagent (Life Technologies) according to the manufacturer's protocol. RNA was either DNased with DNase I recombinant (Roche) and used for cDNA synthesis with iScript™ (Biorad) or was DNased and cDNA synthesised using the iScriPt™ gDNA Clear cDNA Synthesis kit (Biorad). qPCR was done as per standard Biotelliga laboratory protocol using SsoAdvanced™ Universal SYBR Green Supermix (Biorad).

Chemical Analysis

Production of relevant compounds (ACPP, 1-AP, AcAP, and lolines) by transformed fungi were detected with liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The values of detected compounds are represented in micromolar (μM) and were calculated using the following formula:

Concentration in μM=(Concentration in μg/ml±molecular weight)×1000

The molecular weights of the compounds are: 216 (ACPP), 126 (1-AP), 168 (AcAP), 182 (NANL), 154 (loline), and 182 (NFL).

Results

Expression of lolC Results in Production of ACPP in Heterologous Hosts

Of the non-Epichloe fungi tested, A. niger, B. bassiana, M. robertsii, and T. reesei produced ACPP constitutively in culture upon expression of Ef lolC (Table 5). A. niger, B. bassiana, and T. reesei also produced ACPP constitutively in culture upon expression of Pe lolC (Table 5). Pe lolC was not attempted to be transformed into M. robertsii. The ACPP of biological origin was identical to chemically synthesised ACPP. It was observed in typical extracted chromatograms for the protonated mass of ACPP (m/z 217) in transformants with lolC, compared to wild type and a transformant not carrying lolC, but with the same selectable marker (FIG. 4), N. crassa and S. cerevisiae did not produce ACPP although transcription of lolC was detected.

TABLE 5 Summary of observations of expression of lolC and ACPP production in heterologous hosts Summary of transformed Plasmid Transcription ACPP Fungus gene of interest number of lolC detected A. niger lolC pBTL6 Yes 149-322 μM ATCC 1015 (from E. festucae E2368) A. niger lolC pBTL74 Yes 212-913 μM ATCC 1015 (from P. expansum ICMP 8595) B. bassiana lolC pBTL6 Yes 10-1390 μM K4B1 (from E. festucae E2368) B. bassiana lolC pBTL74 Yes 476-1844 μM K4B1 (from P. expansum ICMP 8595) M. robertsii lolC pBTL6 Yes 7-180 μM ARSEF 23 (from E. festucae E2368) N. crassa lolC pBTL6 Yes No ACPP ICMP 7781 (from E. festucae E2368) detected N. crassa lolC Transformed Yes No ACPP ICMP 7781 (from E. festucae E2368, exons as PCR detected only, codon optimized for product N. crassa, C-terminal HA tag) S. cerevisiae lolC pBTL14 No No ACPP BY4743 (from E. festucae E2368, exons detected only, codon optimized for N. crassa,C-terminal HA tag removed) S. cerevisiae lolC pBTL13 Yes No ACPP BY4743 (from E. festucae E2368, exons detected only, codon optimized for S. cerevisiae, C-terminal HA tag) S. cerevisiae lolC (from E. festucae Modified No No ACPP BY4743 E2368, exons only, codon pBTL13 detected optimized for S. cerevisiae, C-terminal HA tag removed) T. reesei lolC pBTL6 Yes 2-17 μM RUT-C30 (from E. festucae E2368) T. reesei lolC pBTL74 Yes 4-878 μM RUT-C30 (from P. expansum ICMP 8595)

Expression of Epichloë Loline Pathway Genes Results in Loline Production in Heterologous Hosts

Based on their ability to produce ACPP upon expression of lolC, heterologous hosts A. niger, B. bassiana, and M. robertsii, along with E. festucae Fl1, were selected as candidate heterologous hosts for expression of lolCDFA1TEOU—or subsets of genes thereof. The lol genes transformed were obtained from E. festucae E2368, E. uncinata AR1006 and/or P. expansum ICMP 8595. The industrial A. niger strain ATCC 1015, was transformed with Epichloë lolD, F, T, A1, O. Cultures were supplemented with 1 mM ACPP, and upon feeding, successfully produced 0.385 μM NANL (Table 6).

Transformation of Epichloë lolCDFA1TEU to B. bassiana resulted in production of 0.179 μM AcAPwhen fed with 2 mM ACPP (see FIGS. 6 and 7 for comparison of AcAP produced by the Epichloë lolCDFA1TEU-carrying transformant Bb CT3 to chemically synthesised AcAP). This may be indicative of the selection of a transformant with relatively weak gene expression, which may have been caused by exclusion of transformants with ‘good’ gene expression due to toxicity of pathway intermediates to the host. Transformation of B. bassiana with Epichloë lolCDFA1TEOU under selected promoters and terminators (see Table 11 for details of constructs) resulted in production of 0.385 μM NANL (see FIG. 8 for chromatogram), as well as the full complement of detectable intermediates post-ACPP (see FIG. 9 for chromatograms). Expression of Epichloë lolCDFA1TEOUMNP in B. bassiana resulted in NFL and loline (see Table 6 for detailed summary of results). Gene expression levels of transformant isolate Bb4-18-H15 (‘Bb H15’) showed possible bottlenecks in the pathway due to relatively ‘low’ expression of lolF and lolO (FIG. 10). lolF and/or lolO, each under E. festucae Fl1 histone H3 promoter, shown to result in relatively high expression levels in our previous experiments, and followed by the g/aA terminator, were transformed into Bb H15. Transformants carrying the additional copy of lolO (named isolate Bb 016) produced more NANL than parent Bb H15. When fed with 30 mM proline, 4 mM OAH, 2 mM alpha-ketoglutaric acid and 0.25 mM iron [in ammoniumiron(II)sulfate hexahydrate (Pan, et al., 2018)], this isolate, Bb016 produced the hightest amount of NANL observed in any heterologous host system to-date (5.49 μM NANL at 4 days post inoculation). Analysis of gene expression of the two isolates showed increased expression in all loline genes in Bb O16 compared to Bb H15 (FIG. 10).

TABLE 6 Summary observations of expression of Epichloë loline pathway genes and lolines and/or intermediates production in hosts tested positive for ACPP with lolC expression Summary of Lolines and/or transformed Plasmid Transcription intermediates Fungus genes of interest number of lol genes detected A. niger Ef lolDFTO, pBTL38, Transcripts of 0.385 μM ATCC Eu lolA1 pBTL32, Ef lolDFTO NANL (in 1015 and Pe lolC pBTL40, detected. cultures fed pBTL57, Pe lolC gene with 1 mM pBTL33 didn't ACPP) integrate. B. Ef lolCDFTEOU pBTL11, Yes, except 0.179 μM bassiana and Eu lolA1 pBTL12 for lolO (in AcAP (in K4B1 pBTL12), cultures fed which had a with 2 mM SNP and ACPP, but thereby not in cultures truncated grown with and non- precursors functional. proline and OAH). See FIGS. 6 and 7 for chromatograms comparing synthetic vs. biologically- produced AcAP. B. Ef lolCDFTEOU pBTL15, Yes 0.385 μM bassiana and Eu lolA1 pBTL16, NANL K4B1 pBTL17 B. Ef pBTL15, Yes 0.055 μM NFL, bassiana lolCDFTEOUMNP pBTL16, 0.065 μM loline K4B1 and Eu lolA1 pBTL17, pBTL18 E. Ef lolDFTEOU pBTL15, Yes 0.22 μM NANL festucae and Eu lolA1 pBTL16, (in cultures fed Fl1 pBTL17 with 1.6 mM ACPP) Expression of P. expansum 101 Genes Result in Loline Production in Heterologous Hosts

Heterologous hosts A. niger and B. bassiana, which were capable of producing ACPP by expression of Pe lolC (Table 5), were transformed with Epichloë lolDFTA1, or P. expansum lolDFTO, henceforth Pe lolDFTO, or subsets of these genes thereof. All genes in transformants were confirmed to be expressed using qRT-PCR. Both A. niger and B. bassiana successfully produced loline pathway intermediates ACPP, 1-AP, and AcAP in the transformant strains expressing the Pe lolCDFTO genes (see Table 7 for details). However, in both A. niger and B. bassiana, transformants did not produce any detectable levels of NANL despite producing relatively high amounts of the precursor intermediate AcAP. This was unexpected as these transformants were expressing Pe lolO ≥1-fold relative to actin, compared to previous transformants which produced detectable levels of NANL with even ≤1-fold expression of Ef lolO (relative to actin). This may indicate that, at least in heterologous systems, Pe lolO is less efficient than Ef lolO in converting AcAP to NANL. Therefore, Ef lolO was transformed to B. bassiana transformants already expressing Pe lolCDFTO gene array. Biological triplicates of 17 transformants that resulted from transformation of Ef lolO to the Pe CDFTO background were screened for Ef lolO and Pe lolO expression levels. From these, 12 transformants with the highest Ef lolO/Pe lolO expression were selected for testing gene expression of the remaining loline genes. (see Table 8 for expression of Pe lolC, D, F, T, O and Ef lolO in biological triplicates of the 12 transformants). Variable gene expression further supported selection against toxic intermediate production in this system and highlighted the requirement for careful selection of transformants with appropriate gene expression ratios. A transformant (no. 17 in Table 8), which did not express Pe lolC, but expressed all five subsequent genes, was fed with 2 mM ACPP and produced 3.63 μM NANL.

TABLE 7 Summary observations of expression of P. expansum lol genes and lolines and/or intermediates production in hosts tested positive for ACPP with Pe lolC expression Summary of transformed Lolines and/or genes of Plasmid Transcription intermediates Fungus interest number of lol genes detected A. niger Pe lolCDFTO pBTL74, Yes 610 μM ACPP, ATCC pBTL78, 450 μM 1-AP, 7.3 1015 pBTL77, μM AcAP (in the pBTL76, transformant pBTL75 with the most loline pathway output); NANL not detected A. niger Pe lolC, Ef pBTL74, Yes 385 μM ACPP, 45 ATCC lolDFT and pBTL38, μM 1-AP, 18 μM 1015 Eu lolA1 pBTL32, AcAP (in the pBTL40, transformant pBTL57 with the most loline pathway output) B. bassiana Pe lolCDFTO pBTL74, Yes 1892 μM ACPP, K4B1 pBTL78, 35 μM 1-AP, 68 pBTL77, μM AcAP (in the pBTL76, transformant pBTL75 with the most loline pathway output); NANL not detected. B. bassiana Pe lolCDFTO pBTL74, Yes 3.63 μM NANL K4B1 and Ef lolO pBTL78, (in a pBTL77, transformant pBTL76, expressing Pe pBTL75, lolDFTO and Ef pBTL33 lolO fed with 2 mM ACPP).

TABLE 8 Gene expression in biological triplicate cultures of B. bassiana transformants positive for Ef lolO in Pe lolC,D,F,T,O parent background Fold change (relative to act) Isolate Triplicate Pe Pe Pe Pe Pe Ef Chemical number ID lolC lolD lolF lolT lolO lolO analysis 5  5a 0.07 13.91 0.23 0.05 3.58 6.61 Not  5b 2.40 11.70 0.36 0.24 2.73 3.88 processed  5c 3.59 19.18 3.58 0.35 2.51 2.84 for   chemical   analysis   since all gene   expressed   but low   expression   of lolF,T. 7  7a 15.24 30.73 0.00 0.00 3.77 3.17 Not  7b 12.94 34.60 0.00 0.00 3.77 3.60 processed  7c 17.15 25.39 0.00 0.00 3.39 4.85 for   chemical analysis   since Pe   lolF, T not   expressed. 8  8a 12.52 34.72 52.31 4.10 3.36 1.49 2142 μM  8b 8.33 2.84 31.88 5.33 4.13 2.53 ACPP, 73  8c 9.03 13.58 41.65 3.16 4.57 3.10 μM 1-AP   and 4 μM AcAP.   NANL not   detected. 9  9a 16.06 12.03 0.00 0.00 3.72 4.56 Not  9b 23.27 9.19 0.00 0.00 6.41 7.00 processed  9c 11.48 37.46 0.02 0.00 4.80 4.01 for   chemical   analysis since Pe lolF, T not expressed. 14 14a 24.59 0.00 5.40 0.00 1.45 15.88 Not 14b 6.77 0.00 6.69 0.00 1.15 5.85 processed 14c 4.96 0.00 5.06 0.00 0.99 4.59 for chemical analysis since Pe lolD, T not expressed. 15 15a 22.36 8.70 0.00 4.47 4.85 8.58 Not 15b 12.44 7.98 0.00 2.65 3.32 6.98 processed 15c 19.75 19.14 0.00 2.44 4.99 7.73 for chemical analysis since Pe lolF not expressed. 17 17a 0.00 21.16 10.04 0.93 4.58 5.11 Only Pe 17b 0.00 29.66 8.85 0.75 4.10 4.90 lolC not 17c 0.00 56.08 30.17 0.82 9.27 4.73 expressed, hence fed 2 mM ACPP. 147 μM 1-AP, 24 μM AcAP and 3.63 μM NANL. 19 19a 11.88 12.05 0.00 0.00 2.82 3.18 Not 19b 26.58 15.92 0.00 0.00 3.97 3.63 processed 19c 21.68 38.78 0.00 0.00 4.35 2.71 for chemical analysis since Pe lolF, T not expressed. 20 20a 0.00 0.00 0.00 0.00 5.36 13.89 Not 20b 0.00 0.00 0.00 0.00 5.20 9.33 processed 20c 0.00 0.00 0.00 0.00 4.96 12.60 for chemical analysis since Pe lolC,D,F, T not expressed. 22 22a 32.89 59.77 24.56 5.50 3.89 5.47 2463 μM 22b 46.22 103.09 44.81 3.54 6.78 3.93 ACPP and 22c 21.07 37.53 11.43 4.05 3.84 4.00 11 μM 1- AP. AcAP and NANL not detected. 24 24a 0.00 0.00 0.00 0.00 11.75 11.41 Not 24b 0.00 0.00 0.00 0.00 4.72 4.82 processed 24c 0.00 0.00 0.00 0.00 10.70 6.46 for chemical analysis since Pe lolC,D,F, T not expressed. 25 25a 24.01 57.00 31.40 0.00 3.66 1.83 Not 25b 48.38 129.60 97.61 0.00 6.61 3.06 processed 25c 31.34 183.34 87.54 0.00 7.87 2.84 for chemical analysis since Pe lolT not expressed.

A Native N-Acetyltransferase of the Heterologous Host Converts 1-AP to AcAP

Epichloë lolCDFTEOUA1 were transformed to heterologous hosts in accordance with the general scientific consensus that the former seven genes from Epichloë are necessary and sufficient to produce NANL, and that lolA, while not necessary for NANL production, would increase precursor OAH levels resulting in a good flux of intermediates through the pathway. However, existing experimental evidence does not fully clarify the function and necessity of lolD, lolF, lolU, and lolE for NANL production (see Table 9 for summary and references to experimental evidence of loline gene functions).

TABLE 9 Putative functions of loline pathway genes and their role in the loline pathway. Predicted function (as listed on Putative role of the Reference for Gene (Schardl, et al., encoded enzyme in experimental name 2013)) the loline pathway evidence loLA Amino acid Increasing the levels of Current study binding OAH lolC gamma-class PLP Formation of ACPP Current study enzyme from proline and OAH lolD alpha-class PLP Decarboxylation of Not available enzyme pyrrolodinium ion lolF Monooxygenase Oxidative Not available decarboxylation of ACPP to form pyrrolodinium ion lolT alpha-class PLP Cyclisation of (Zhang, et al., 2009, enzyme pyrrolodinium Pan, et al., 2014) ion(s) to form 1-AP lolE Nonheme iron Not clear Not available dioxygenase lolO Nonheme iron Formation of the C2-C7 (Pan, et al., 2014, dioxygenase ether bridge to form Pan, et al., 2018) NANL lolU Unknown Unknown Not available lolN Acetannidase Deacetylation of NANL (Pan, et al., 2014) to form norloline lolM N- Methylation of norloline (Pan, et al., 2014) Methyltransferase to form loline, and of loline to form NML lolP Cytochrome P450 Oxygenation of NML to (Spiering, et al., monooxygenase form NFL 2008) Note only the references which give experimental proof of gene function are listed.

The roles of lolF and lolD are well supported by biochemical theory that the pyrrolodinium ions, which are the putative products of LoID- and LoIF-catalysed reactions, are likely to be highly unstable compounds not able to be synthesised for authentic standards or detected by targeted LC-MS/MS (D. Rennison, personal communication, 2016). Therefore, the applicants focused on clarifying the function of lolE and lolU.

lolU was considered by the applicants to be a possible candidate for the acetylation of 1-AP to AcAP, based on the identification of an HMM signature of CoA-dependent acyltransferases superfamily in InterPro (https://www.ebi.ac.uk/interpro/) and the top hit to acetyltransferase in Swiss-MODEL structural analysis (https://swissmodel.expasy.org/). When fed with 2 mM 1-AP, AcAP was detected in two independent B. bassiana transformants which were expressing E. festucae E2368 lolU, as well as in two independent transformants without lolU. No AcAP was observed in the growth medium with 2 mM 1-AP control. No statistical difference in 1-AP acetylation was observed between lolU-containing and non-containing B. bassiana K4B1. To further explore this preliminary data that showed that an N-acetyltransferase gene native to the host is capable of the acetylation of 1-AP to AcAP, a 1-AP feeding assay was done using a range of fungi. No ACPP or hydroxy-AcAP was detected in any culture. However, wild type strains of all fungi tested, except for E. festucae Fl1, S. cerevisiae and S. indica, were capable of acetylating 1-AP to AcAP (see Table 10 for details). All other Epichloë strains tested (E. uncinata AR1006 and E. festucae E2368), and an Fl1 strain carrying Ef lolDFTEOU were capable of the acetylation however, which may indicate that the Fl1 strain tested may have converted 1-AP to AcAP, albeit less efficiently—and thus at levels less than the detection threshold. While the ability of wild type fungi to convert 1-AP to AcAP without lolU is not conclusive evidence that lolU plays a role in acetylation of 1-AP to AcAP in the native system, it is proof that a native acetyl-transferase, perhaps universally present across Kingdom Fungi, has the ability to convert 1-AP to AcAP.

TABLE 10 Production of 1-AP to AcAP by different fungi Species Strain 1-AP fed (μM) AcAP detected (μM) A. niger ATCC 1015 1000 10.99 A. niger ATCC 1015 1000 7.15 B. bassiana K4B1 1000 9.97 B. bassiana K4B1 1000 8.63 B. bassiana K4B1 lolU3 1000 8.52 B. bassiana K4B1 lolU3 1000 15.45 B. bassiana K4B1 lolU20 500 9.03 B. bassiana K4B1 lolU20 500 6.28 E. festucae Fl1 1000 <0.25 E. festucae Fl1 1000 <0.25 E. festucae E2368 500 2.38 E. festucae E2368 500 0.48 E. festucae Fl1 l0l9 500 0.95 E. festucae Fl1 l0l9 500 1.41 E. uncinata AR1006 500 0.88 E. uncinata AR1006 500 0.88 E. nigrum SF7849 1000 11.57 E. nigrum SF7850 1000 6.79 P. expansum ICMP 8595 1000 8.11 P. expansum ICMP 8595 1000 5.41 R. solani ICMP 17586 500 7.92 R. solani ICMP 17586 500 7.16 Rhizopus sp. 1000 8.1 Rhizopus sp. 1000 14.58 S. zeae EBTL 218 500 22.94 S. zeae EBTL 219 500 19.22 T. reesei ATCC 56765 1000 32.44 T. reesei ATCC 56765 1000 28.86 S. indica ATCC 204458 1000 <3 S. indica ATCC 204458 1000 <3 U. isabellina ICMP 22148 1000 70 U. isabellina ICMP 22148 1000 82 K. marxianus Y-1008 1000 310 K. marxianus Y-1008 1000 224 S. cerevisiae BY4743 1000 <3 S. cerevisiae BY4743 1000 <3

Epichloë lolE, the only other nonheme iron dioxygenase besides lolO present in the Clavicipitaceae loline cluster, is suggested to be ‘not absolutely required’ in the ether bridge formation due to unpublished observations with a lolE knockout mutant (alluded to in (Pan, et al., 2014)). However, recently it was reported that LolE has no role in vitro in the two oxygenation steps required to form NANL from AcAP (Pan, et al., 2018). But, there is no published data to provide conclusive evidence to whether LolE plays a role in vivo in one of the two oxygenation steps that are required to form NANL from AcAP. To this end, B. bassiana transformants expressing either lolE, lolO, or lolE and lolO together (‘lolEO’) were obtained, fed with 0.8 mM 1-AP, and the mycelial fraction was analysed for compounds of interest. All transformants as well as wild type B. bassiana fed with 1-AP produced AcAP. However, the intermediate generated by hydroxylation of AcAP (hydroxy-AcAP) and NANL were only detected in lolO and lolEO transformant cultures. No AcAP, hydroxy-AcAP, or NANL was detected in the growth medium with 0.8 mM 1-AP control. There was no correlation of lolE expression with hydroxy-AcAP levels in lolEO cultures (R²=0.0009).

Discussion

The study of the current Example sought to achieve three main goals: (1) produce ACPP in heterologous fungal hosts by expression of the lolC gene; (2) produce NANL in heterologous hosts selected due to their ability to form ACPP; and (3) understand the requirement for lolE and lolU in the Clavicipitaceae loline production pathway.

When attempting to produce ACPP by expression of Epichloë lolC, the applicants observed that the native E. festucae gene with the introns and exons expressed under a constitutive promoter consistently resulted in a detectable lolC transcript in all filamentous fungi tested. Transcription of the gene led to ACPP production in culture in B. bassiana, M. robertsii, and the industrial strains A. niger and T. reesei (E. festucae Fl1 was not tested with the E. festucae lolC cassette). Although transcripts were detected in N. crassa, no ACPP was detected. The modified lolC—i.e., Ef lolC exons only, codon-optimzed for N. crassa—resulted in variable levels of transcription in the tested hosts and was unsuccessful in producing ACPP in all cases. The highest amount of ACPP observed in a heterologous host expressing only Ef lolC was 1390 μM, produced by a B. bassiana transformant. Expression of Pe lolC was attempted only in A. niger, B. bassiana, and T. reesei, and resulted in successful transcription of Pe lolC and ACPP production in all three cases. The highest amount of ACPP observed in a heterologous host expressing Pe lolC only was 1844 μM, produced by a B. bassiana transformant. The maximum amount of ACPP observed in any transformant to-date is 2463 μM, which was produced by a B. bassiana transformant carrying Pe lolCDFTO and Ef lolO. It is noteworthy that endogenous ACPP amounts ≥3 mM has not been observed in any heterologous host tested so far. This may be due to the dose-dependent cytotoxic effect of ACPP, which has been reported for E. uncinata in previous literature (Faulkner, et al., 2006).

The applicants successfully produced NANL-loline-NFL, and NANL by itself, as well as the full array of chemically detectable pathway intermediates in B. bassiana via expression of Epichloë lolCDFA1TEOUMNP and Epichloë lolCDFA1TEOU, respectively. To the best of the applicant's knowledge, this is the first report of heterologous production of the full loline pathway in a non-native host. NANL was also produced in transformants expressing Epichloë lolDFTA10 in the industrial A. niger strain ATCC 1015, those expressing Ef lolDFTEOU in E. festucae strain Fl1, and those expressing the Pe lolDFTO-Ef lolO combination in B. bassiana strain K4B1. The latter three cultures were all fed 1 or 2 mM ACPP as they all lacked lolC. All detectable loline pathway intermediates upto NANL, were produced by A. niger and B. bassiana transformants that were expressing Pe lolCDFTO as well, although detectable levels of NANL was absent. The Epichloë loline genes transformed to the heterologous hosts originated from E. uncinata AR1006 (Eu lolA1) and/or E. festucae E2368 (Ef lolCDFTEOUMNP), which has currently been observed to produce lolines in planta only. All Penicillium lol genes are from P. expansum, the only Penicillium species in which the lolgenes have been reported to-date. To the best of the applicant's knowledge, the current study is also the first report of successful heterologous expression of any Pe lol gene.

Analysis of loline gene expression data showed possible bottlenecks in expression of genes such as lolF and loll). Re-transformation of lolO under the E. festucae Fl1 histone H3 promoter that was previously seen to produce relatively ‘high’ gene expression, increased the NANL level from 0.385 μM in Bb H15 to 1.209 μM in Bb O16. A further increase in production in the BbO16 strain was achieved by feeding with iron (in the form of ammoniumiron(II)sulfate hexahydrate) and 2-oxoglutarate, which have been shown to bias the oxygenation reaction catalyzed by lolO-encoded enzyme towards NANL (Pan, et al., 2018).

The applicants saw that both wild type B. bassiana without any loline genes and B. bassiana lolU transformants accumulated AcAP when fed with 1-AP. This is consistent with the previous observation that there was no obvious change in the loline alkaloid profile of lolU RNAi transformants (Pan, 2014). Therefore, the applicants conclude that lolU, although a putative acetyl transferase, is not exclusively responsible for the acetylation of 1-AP to AcAP, and that a native gene of the heterologous host is capable of this conversion. The applicants also observed no lolU expression in transformant Bb O16, although it produced NANL. Collectively, these observations make the necessity of lolU in the loline pathway questionable.

Production of NANL in A. niger ATCC 1015 strain is of significance as it is a strain well-suited- and commonly used—in fermentation industry. B. bassiana, the heterologous host which has produced the highest levels of lolines in the systems tested so far, is reported as a generalist systemic endophyte. Suitability when using as a spray treatment, lifespan in soil vs. foliage, effect on plant growth, and ability to form a loline-producing but non-sporulating mutant all remain basic questions to be answered.

Based on our observations, a two-step screen, which consists of checking the ability of a selected fungus to (1) produce ACPP through expression of lolC and (2) convert 1-AP to AcAP without any transgenes, is proposed when selecting a heterologous host for expression of lolines. If a fungus is capable of both, and is fermentation compatible and/or is an established endophyte, it may be an optimal heterologous host for loline production. The best experimental approach should be then determined based on the ACPP- and 1-AP-feeding experiment results and availability of selectable markers for the selected fungus. If a fungus with a high potential as an endophyte or fermentation compatibility is capable of producing ACPP, but is unable to convert 1-AP to AcAP, it may be useful to check if it could accumulate ‘high’ levels of 1-AP. If so, it may be used in a sequential fermentation, where the 1-AP formed is converted to the next products of the pathway by a co-cultivated host. Alternatively, if a gene that can convert 1-AP to AcAP is identified, it can be expressed in the fungus along with the other loline genes, thus giving it the ability to make the desired end product. On the other hand, if a fungus is capable of the 1-AP to AcAP conversion, but cannot produce ACPP, it may be useful to determine if ACPP-toxicity has led to the mortality of ACPP-producers and if so, an approach which transforms other desired loline genes prior to transformation of lolC, and transformation of lolC last could be considered. A case-by-case review of risks vs. benefits for each scenario is recommended.

Following the experiments of this example, the applicants postulate that, contrary to previous expectation for requirement of seven genes, expression of just five genes —lolC, lolD, lolF, lolT, lolO—in a heterologous host should result in the production of NANL—the first fully cyclized loline intermediate of the pathway.

TABLE 11 Detail of loline gene constructs used in the current application Alternative Transgene Promoter loline (Predicted) Name name promoter(s) organism gene Gene function Gene origin Gene modifications pBTL2 pBAR-GFP Translation Aureobasidium None Not applicable Not applicable Not applicable elongation factor pullulans 1a pBTL4 pUC57- Glyceraldehyde 3- M. robertsii ARSEF lolC γ-class PLP E. festucae Codon-optimised for N. lolCDF phosphate 23 cystathionine E2368 crassa, CDS, C terminal synthase HA tag lolD PLP-containing E. festucae Codon-optimised for N. ornithine E2368 crassa, CDS, C terminal decarboxylase HA tag lolF FAD-containing E. festucae Codon-optimised for N. monooxygenase E2368 crassa, CDS, C terminal HA tag pBTL5 pBargfp- Glyceraldehyde 3- M. robertsii ARSEF lolC γ-class PLP E. festucae Codon-optimised for N. lolCDF phosphate 23 cystathionine E2368 crassa, CDS, C terminal synthase HA tag Glyceraldehyde 3- M. robertsii ARSEF lolD PLP-containing E. festucae Codon-optimised for N. phosphate 23 ornithine E2368 crassa, CDS, C terminal decarboxylase HA tag Glyceraldehyde 3- M. robertsii ARSEF lolF FAD-containing E. festucae Codon-optimised for N. phosphate 23 monooxygenase E2368 crassa, CDS, C terminal HA tag pBTL6 pH3-lolC Histone H3 E. festucae Fl1 lolC γ-class PLP E. festucae none cystathionine E2368 synthase pBTL8 pChuk4:lolC TDH3 (same as S. cerevisiae lolC γ-class PLP E. festucae Codon-optimised for N. glyceraldehyde 3- BY4743 cystathionine E2368 crassa, CDS phosphate) synthase pBTL9 pUC57- Translation E. festucae E2368 lolT PLP-containing E. festucae T2A peptides between T2A::TEO elongation factor pyrolizidinase E2368 lolT and lolE, and 1a between lolE and lolO.Codon-optimized for N. crassa, CDS Translation E. festucae E2368 lolE oxidoreductase E. festucae T2A peptides between elongation factor E2368 lolT and lolE, and 1a between lolE and lolO.Codon-optimized for N. crassa, CDS Translation E. festucae E2368 lolO non-heme iron E. festucae T2A peptides between elongation factor oxygenase E2368 lolT and lolE, and 1a between lolE and lolO.Codon-optimized for N. crassa, CDS pBTLTOPO1 TOPO Translation lolT PLP-containing E. festucae Codon-optimised for N. synthetic lolT elongation factor pyrolizidinase E2368 crassa, CDS gblock 1a pBTLTOPO2 TOPO Translation lolE oxidase E. festucae Codon-optimised for N. synthetic lolE elongation factor E2368 crassa, CDS gblock 1a pBTLTOPO3 TOPO Translation lolO non-heme iron E. festucae Codon-optimised for N. synthetic lolO elongation factor oxygenase E2368 crassa, CDS gblock 1a WT-DFA Glyceraldehyde 3- M. robertsii ARSEF lolD PLP-containing E. festucae none phosphate 23 ornithine E2368 decarboxylase Glyceraldehyde 3- E. festucae Fl1 lolF FAD-containing E. festucae none phosphate monooxygenase E2368 Histone H3 E. festucae Fl1 lolAl Amino acid E. uncinata CDS binding AR1006 pBTL11 WT-CDFA Translation M. robertsii ARSEF lolC γ-class PLP E. festucae none elongation factor 23 cystathionine E2368 1a synthase Glyceraldehyde 3- M. robertsii ARSEF lolD PLP-containing E. festucae none phosphate 23 ornithine E2368 decarboxylase Glyceraldehyde 3- E. festucae Fl1 lolF FAD-containing E. festucae none phosphate monooxygenase E2368 Histone H3 E. festucae Fl1 lolAl Amino acid E. uncinata CDS binding AR1006 pBTL12 WT-TEOU Hexokinase-1 M. robertsii ARSEF lolT PLP-containing E. festucae none 23 pyrolizidinase E2368 Histone H3 M. robertsii ARSEF lolE oxidase E. festucae none 23 E2368 Glyceraldehyde 3- A. nidulans lolO non-heme iron E. festucae Contains SNP phosphate oxygenase E2368 Translation M. robertsii ARSEF lolU 15-O- E. festucae none elongation factor 23 acetyltransferase E2368 1a pBTL13 pChuk4: lolC TDH3 (same S. cerevisiae lolC γ-class PLP E. festucae Codon-optimised for s. (Sc, with HA glyceraldehyde 3- BY4743 cystathionine E2368 cerevisiae, CDS, C tag) phosphate) synthase terminal HA tag pBTL14 pChuk4: lolC TDH3 (same S. cerevisiae lolC γ-class PLP E. festucae Codon-optimised for N. (Sc, without glyceraldehyde 3- BY4743 cystathionine E2368 crassa, CDS HA tag) phosphate) synthase pBTL15 pCA Translation M. robertsii ARSEF lolA1 Amino acid E. uncinata CDS elongation factor 23 binding AR1006 1a Histone H3 E. festucae Fl1 lolC γ-class PLP E. festucae none cystathionine E2368 synthase pBTL16 pDFT Histone H3 E. festucae Fl1 lolD PLP-containing E. festucae none ornithine E2368 decarboxylase Histone H3 B. bassiana K4B1 lolF FAD-containing E. festucae none monooxygenase E2368 Translation M. robertsii ARSEF lolT PLP-containing E. festucae none elongation factor 23 pyrolizidinase E2368 1a pBTL17 pEOU Histone H3 E. festucae Fl1 lolE oxidase E. festucae none E2368 Histone H3 B. bassiana K4B1 lolO non-heme iron E. festucae none oxygenase E2368 Translation M. robertsii ARSEF lolU 15-O- E. festucae none elongation factor 23 acetyltransferase E2368 1a pBTL18 pMNP Histone H3 E. festucae Fl1 lolM N- E. festucae none methyltransferase E2368 Histone H3 B. bassiana K4B1 lolN acetamidase E. festucae none E2368 Translation M. robertsii ARSEF lolP cytochrome P450 E. festucae none elongation factor 23 E2368 1a pBTL32 plolFnew Histone H3 E. festucae Fl1 lolF FAD-containing E. festucae none monooxygenase E2368 pBTL33 plolOnew Histone H3 E. festucae Fl1 lolO non-heme iron E. festucae none oxygenase E2368 pBTL38 plolD Histone H3 E. festucae Fl1 lolD PLP-containing E. festucae none ornithine E2368 decarboxylase pBTL40 plolT Translation M. robertsii ARSEF lolT PLP-containing E. festucae none elongation factor 23 pyrolizidinase E2368 1a pBT7L55 lolU Class I B. bassiana K4B1 lolU 15-O- E. festucae none overexpression Hydrophobin acetyltransferase E2368 pBTL56 lolO Class I B. bassiana K4B1 lolO non-heme iron E. festucae none overexpression Hydrophobin oxygenase E2368 pBTL57 plolA Translation M. robertsii ARSEF lolA1 Amino acid E. uncinata CDS elongation factor 23 binding AR1006 1a pBTL58 plolC Histone H3 E. festucae Fl1 lolC γ-class PLP E. festucae none cystathionine E2368 synthase pBTL59 plolE Histone H3 E. festucae Fl1 lolE oxidase E. festucae none E2368 pBTL60 plolO Histone H3 B. bassiana K4B1 lolO non-heme iron E. festucae none oxygenase E2368 pBTL61 plolU Translation M. robertsii ARSEF lolU 15-O- E. festucae none elongation factor 23 acetyltransferase E2368 1a pBTL62 plolM Histone H3 E. festucae Fl1 lolM N- E. festucae none methyltransferase E2368 pBTL63 plolN Histone H3 B. bassiana K4B1 lolN acetamidase E. festucae none E2368 pBTL64 plolP Translation M. robertsii ARSEF lolP cytochrome P450 E. festucae none elongation factor 23 E2368 1a pBTL71 pFI1H3/lolT/ Histone H3 E. festucae Fl1 lolT PLP-containing E. festucae none glaA pyrolizidinase E2368 pBTL74 pPe-lolC Histone H3 E. festucae Fl1 lolC γ-class PLP P. expansum none cystathionine ICMP 8595 synthase pBTL75 pPe-lolO Histone H3 E. festucae Fl1 lolO non-heme iron P. expansum none oxygenase ICMP 8595 pBTL76 pPe-lolT Histone H3 E. festucae Fl1 lolT PLP-containing P. expansum none pyrolizidinase ICMP 8595 pBTL77 pPe-lolF Histone H3 E. festucae Fl1 lolF FAD-containing P. expansum none monooxygenase ICMP 8595 pBTL78 pPe-lolD Histone H3 E. festucae Fl1 lolD PLP-containing P. expansum none ornithine ICMP 8595 decarboxylase pBTL80 P_(PIGPD)-hph- Translation S. indica lolC γ-class PLP P. expansum none ter_(glaA) elongation factor cystathionine ICMP 8595 1a synthase pBTL81 WT-TEOU Hexokinase-1 M. robertsii ARSEF lolT PLP-containing E. festucae none 23 pyrolizidinase E2368 Histone H3 M. robertsii ARSEF lolE oxidase E. festucae none 23 E2368 Glyceraldehyde 3- A. nidulans lolO non-heme iron E. festucae none phosphate oxygenase E2368 Translation M. robertsii ARSEF lolU 15-O- E. festucae none elongation factor 23 acetyltransferase E2368 1a

Example 3: Screening Multiple Taxa for their Ability to Endogenously Generate AcAP from 1-AP Introduction/Summary

The protein encoded by lolU was considered a possible candidate for the acetylation of 1-AP to AcAP, based on its structural similarity to N-acetyltransferases. However, when fed with 2 mM 1-AP, AcAP was detected in two independent B. bassiana transformants which were expressing E. festucae E2368 lolU, as well as in two independent transformants without lolU. No AcAP was observed in the growth medium with 2 mM 1-AP control. This indicated that an N-acetyltransferase gene native to the host was capable of the acetylation of 1-AP to AcAP. As this gene is yet unidentified, the ability to convert 1-AP to AcAP is currently a required characteristic for a given fungus to successfully produce NANL. Therefore, a screen of multiple fungal taxa for the ability to convert 1-AP to AcAP was proposed. This screen has been done with a set of fungi from phylum Ascomycota, to which the native loline producer Epichloë species belong, and a fungus from unplaced subphylum Mucoromycota (formerly Zygomycota). All strains tested, except Epichloë festucae strain Fl1 were able to acetylate 1-AP to form AcAP.

Materials and Methods Fungal Strains

All fungal strains (Table 12) were sub-cultured onto potato dextrose agar (PDA) from original or glycerol stocks where possible. All subsequent sub-cultures were also performed with PDA.

TABLE 12 Fungal strains used in this study No. Species name Strain Class Source  1 Aspergillus niger ATCC 1015 Eurotiomycetes U.S.A  2 Beauveria bassiana K4B1 Sordariomycetes New Zealand  3 Beauveria bassiana K4B1 ::lolU3 Sordariomycetes New Zealand  4 Beauveria bassiana K4B1 ::lolU20 Sordariomycetes New Zealand  5 Epichlo{umlaut over (e)} festucae Fl1 Sordariomycetes U.S.A  6 Epicoccum sp. SF7849 Dothidiomycetes New Zealand  7 Metarhizium robertsii ARSEF 23 Sordariomycetes U.S.A  8 Penicillium expansum ICMP 8595 Eurotiomycetes Spain  9 Rhizoctonia solani ICMP 17586 Agaricomycetes New Zealand 10 Rhizopus sp. Mucoromycotina New Zealand 11 Sarocladium spp. BTL-E218 Sordariomycetes New Zealand 12 Trichoderma reesei ATCC 56765 Sordariomycetes U.S.A

Establishing Optimal Media for all Fungal Strains

A preliminary growth study for six wild type strains of fungi was performed using two complex media, PD broth (PDB) and sabouraud dextrose broth (SDB). The strains used were A. niger, B. bassiana, E. festucae, M. robertsii, P. expansum, and T. reesei.

A small square of fungi (approximately 0.5 cm²) was added to 1 ml of MilliQ water in a sterile bead beating tube. The mycelial suspension was macerated in a tissue homogenizer for 30 seconds at 4,000 RPM. 40 μl of this mycelium was then added to four 50 ml Falcon tubes containing 2 ml of SDB or PDB in duplicate.

After one day the cultures were observed and growth was recorded. Those with significant biomass were fed with 22 μl of 60% w/v ethanol. Three days post-inoculation, the cultures were again observed and growth noted. Overall growth was measured using a subjective scale.

Fungal Cultures and Inoculation

All fungi, excluding numbers 5 and 7, were inoculated in batches, based on qualitative observations of their growth. There are three groups, with fast, medium, and slow growing species. Each fungus was correspondingly inoculated so that the feeding of 1-AP could all be performed at the same time for all species. A small square of fungi approximately 1 cm²) was added to 1 ml of MilliQ water in a sterile bead beating tube. This tube was then bead beaten in a tissue homogenizer for 30 seconds at 4,000 RPM. 40 μl of this mycelium was then added to five 50 ml Falcon tubes containing 2 ml of SDB; duplicate treatment and triplicate treatment cultures. The lids were loosely fitted on the tubes and secured with sellotape. These tubes were then incubated in an upright rack at 25° C. with shaking at 125 RPM in the dark. Four media-only controls were also incubated in these conditions.

Feeding of 1-AP

1-AP solution sufficient for the required number of treatments to give a final concentration of 2 mM was run through a vacuum pump for three minutes to concentrate it, leaving a solution containing roughly 60% w/v ethanol. This 1-AP solution was then fed in equal amounts to the treatment cultures, and an equivalent volume of 60% w/v ethanol added to the control tubes. All tubes were incubated in the same conditions for 48-72 h before harvesting.

Harvesting of Chemical Samples

One ml of mycelium and broth was macerated by bead-beating in a Precellys homogenizer, then filtered through a 0.2 μm syringe filter. Filtrates were then analysed for loline intermediates via LC-MS/MS.

Biomass Measurements

For each control tube, the dry biomass was determined; the filter papers were allowed to incubate at room temperature for several days before weighing.

Results Biomass Measurement

Individual weights of a set of labelled filter papers were recorded. Mycelium was sterilized holding ≥20 min in 10% Prevail®, and was added to the corresponding filter paper and was incubated at 60° C., overnight. As some of these papers had brown charred sections were lighter than expected, they were left on the bench for three more days and were weighed again. The average dry biomass weight for all species ranged between 22 and 52 mg. The highest biomass was produced by A. niger ATCC 1015, and the lowest by E. festucae Fl1 and Rhizopus spp.

Chemistry Analysis

Initially, eight different strains were analysed to test for their endogenous ability to convert 1-AP to AcAP. Of these eight, seven were found to be able to perform this conversion, with the exception being E. festucae Fl1. Roughly 0.5-3 percent of 1-AP was converted to AcAP. The highest average amount of AcAP measured was 267 μM by for K. marxianus; and the lowest was 9.1 μM by A. niger ATCC 1015 (Table 10).

Chemistry samples for B. bassiana K4B1 lolU #20, R. solani ICMP 17586, and Sarocladium spp. BTL-E218 were harvested, but not analysed.

Discussion

In the current screen conducted, all fungal species tested, except E. festucae Fl1 strain, were capable of acetylating 1-AP to AcAP. The amount of AcAP produced by different strains was low but still highly variable among species, with the production of AcAP by T. reesei in this study the highest detected from any 1-AP feeding study or produced by heterologous hosts in any instance (FIG. 11). This preliminary analysis supports the utility of a screen for 1-AP acetylation in identifying a suitable heterologous host for loline production.

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INDUSTRIAL APPLICATION

The expression constructs, host cells, and methods of the invention have utility for many agricultural, horticultural, medical and veterinary applications, such as providing horticulturalists with a useful means of controlling plant pests, and providing therapies for the treatment or prevention of insect infection or infestation in humans or non-human animals.

-   -   Claims 1-19 (canceled) 

20. A host cell able to produce more of at least one loline alkaloid or precursor thereof, than does a control cell, as a result of the host cell being transformed or modified to comprise at least one polynucleotide selected from the group consisting of: i) a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:1, 12, 13 and 61 or a variant thereof with at least 40% identity to any one of SEQ ID NO:1, 12, 13 and 61 with at least one of activity of a gamma-class PLP enzyme and an activity substantially equivalent to that of a lolC gene product, ii) a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:2, 14, 15 and 62 or a variant thereof with at least 40% identity to any one of SEQ ID NO:2, 14, 15 and 62 with at least one of activity of an alpha-class PLP enzyme and activity substantially equivalent to that of the lolD gene product, iii) a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:3, 16, 17 and 63 or a variant thereof with at least 40% identity to of any one of SEQ ID NO:3, 16, 17 and 63 with at least one of monooxygenase activity and activity substantially equivalent to that of the lolF gene product, iv) a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:4, 18 and 19 or a variant thereof with at least 40% identity of any one of SEQ ID NO:4, 18 and 19 with at least one of amino acid bridging activity and activity substantially equivalent to that of the lolA gene product, v) a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:5, 20, 21 and 64 or a variant thereof with at least 40% identity to of any one of SEQ ID NO:5, 20, 21 and 64 with at least one of activity of an alpha-class PLP enzyme and activity substantially equivalent to that of the lolT gene product, vi) a polynucleotide encoding a polypeptide comprising the sequence of of any one of SEQ ID NO:6, 22, 23 and 65 or a variant thereof with at least 40% identity to of any one of SEQ ID NO:6, 22, 23 and 65 with at least one of activity of a non-heme iron dioxygenase and activity substantially equivalent to that of the lolE gene product, vii) a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:7, 24, 25 and 66 or a variant thereof with at least 40% identity to any one of SEQ ID NO:7, 24, 25 and 66 with at least one of activity of a non-heme iron dioxygenase and activity substantially equivalent to that of the lolO gene product, viii) a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:8, 26 and 27 or a variant thereof with at least 40% identity to any one of SEQ ID NO:8, 26 and 27 with activity substantially equivalent to that of the lolU gene product, ix) a polynucleotide encoding a polypeptide comprising the sequence of SEQ ID NO:9 or 28 or a variant thereof with at least 40% identity to SEQ ID NO:9 or 28 with at least one of N-Methyltransferase activity and activity substantially equivalent to that of the lolM gene product, x) a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:10, 29 and 67 or a variant thereof with at least 40% identity to any one of SEQ ID NO:10, 29 and 67 with at least one of acetamidase activity and activity substantially equivalent to that of the lolN gene product, and xi) a polynucleotide encoding a polypeptide comprising the sequence of SEQ ID NO:11 or 30 or a variant thereof with at least 40% identity to SEQ ID NO:11 or 30 with at least one of cytochrome P450 monooxygenase activity and activity substantially equivalent to that of the lolP gene product.
 21. The host cell of claim 20 wherein the host cell is able to produce more of at least one loline alkaloid, than does a control cell, as a result of the host cell being transformed or modified to comprise the at least one polynucleotide.
 22. The host cell of claim 20 wherein the host cell is tolerant of endogenous (3-amino-3-carboxypropyl) proline (ACPP) production.
 23. The host cell of claim 22 wherein the host cell has been pre-selected for tolerance to cellular ACPP.
 24. The host cell of claim 20 wherein the host cell prior to modification or transformation, is able to convert exo-1-aminopyrrolizidine (1-AP) to exo-1-acetamido-pyrrolizidine (AcAP).
 25. The host cell of claim 24 wherein the host cell prior to modification or transformation, has been pre-selected for the ability to convert 1-AP to AcAP.
 26. The host cell of claim 20 wherein the host cell is transformed or modified to comprise at least the polynucleotides of i), ii), iii), v) and vii).
 27. The host cell of claim 20 wherein the host cell is not transformed or modified to comprise the polynucleotide of vi).
 28. The host cell of claim 20 wherein the host cell is not transformed or modified to comprise the polynucleotide of viii).
 29. A method for producing a host cell that produces at least one loline alkaloid or precursor thereof, the method comprising modifying or transforming a host cell to comprise at least one polynucleotide as defined in claim
 20. 30. The method of claim 29 wherein the host cell produces more of at least one loline alkaloid, than does a control cell, as a result of the host cell being transformed or modified to comprise the at least one polynucleotide.
 31. The method of claim 29 wherein the host cell is tolerant of endogenous (3-amino-3-carboxypropyl) proline (ACPP) production.
 32. The method of claim 29 further comprising the step of pre-selecting the host cell for tolerance to cellular ACPP.
 33. The method of claim 29 wherein the host cell prior to modification or transformation, is able to convert exo-1-aminopyrrolizidine (1-AP) to exo-1-acetamido-pyrrolizidine (AcAP).
 34. The method of claim 29 wherein the host cell prior to modification or transformation, has been pre-selected for the ability to convert 1-AP to AcAP.
 35. The method of claim 29 further comprising the step of pre-selecting the host cell for said ability to convert 1-AP to AcAP.
 36. The method of claim 29 wherein the host cell is transformed or modified to comprise at least the polynucleotides of i), ii), iii), v) and vii).
 37. A method for producing at least one loline alkaloid or a precursor thereof, the method comprising culturing the host cells of claim 1 under conditions conducive to the production of the at least one loline alkaloid or precursor thereof, by the host cells.
 38. The method of claim 37 wherein the host cell produces at least one loline alkaloid. 